JP2023552846A - Antibodies that bind to metapneumovirus F-protein and uses thereof - Google Patents

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that bind to the F protein (fusion protein) of metapneumovirus (MPV). The antibodies and antigen-binding fragments thereof neutralize MPV infection. The invention also relates to nucleic acids encoding such antibodies and antigen-binding fragments, and cells expressing such antibodies and antibody fragments. Furthermore, the invention relates to methods for detecting and testing MPV antigens and the use of antibodies and antibody fragments in the diagnosis, treatment and prevention of MPV infection.

Description

Detailed description of the invention

The present invention relates to the field of antibodies against metapneumovirus (MPV), in particular antibodies that bind to the F protein (fusion protein) of metapneumovirus (MPV) in its prefusion conformation. The invention also relates to the use of such antibodies, for example in methods for neutralizing MPV infection, for detecting MPV antigens, or for testing MPV vaccines.

Human metapneumovirus (hMPV) was first isolated in 2001 and is a common cause of bronchiolitis and pneumonia in children and the elderly. MPV also causes repeated infections, including severe lower respiratory tract disease, which can occur at any age, especially among the elderly or people with compromised heart, lungs, or immune systems. MPV is associated with 5% to 40% of respiratory tract infections in hospitalized and outpatient children. Infection with MPV is a significant burden of disease in at-risk preterm infants, chronic lung disease, congestive heart disease, and immunodeficiency (Martino et al., 2005, Biology of Blood and Marrow Transplantation: Journal of the American Society for Blood and Marrow Transplantation 11:781-796).

MPV belongs to the genus Metapneumovirus in the subfamily Pneumoviriniae and the family Paramyxoviridae, and is an enveloped non-segmented negative-strand RNA virus. The genetic structure of MPV is similar to that of respiratory syncytial virus (RSV), but MPV lacks the nonstructural genes NS1 and NS2 found in RSV. The RSV and MPV envelopes contain three virus-encoded transmembrane surface glycoproteins: major attachment protein G, fusion protein F, and small hydrophobic SH protein.

The MPV F protein induces viral penetration by fusion between the virion envelope and the plasma membrane of the host cell. Initially, the MPV F protein is expressed as a polypeptide precursor ("F ₀ "). The F protein precursor F ₀ is proteolytically processed at a conserved cleavage site, resulting in F ₁ and F ₂ polypeptides. The mature trimeric F protein is formed by the aggregation of three protomers of the F ₂ -F ₁ heterodimer and assumes a metastable prefusion conformation. The N-terminus of the F subunit is generated by proteolytic cleavage and contains a hydrophobic stretch of amino acids called the fusion peptide, which can be inserted directly into the target membrane to initiate fusion. After binding to the target cell and subsequent activation, the metastable prefusion F protein undergoes a series of structural rearrangements that lead to fusion of the viral and target cell membranes, resulting in the formation of the stable postfusion F protein. undergo rearrangement).

The MPV F protein is the primary target of neutralizing antibodies against MPV and is a target for vaccine development. However, despite the identification of potent neutralizing antibodies such as MPE8 (Corti et al., 2013, Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501: 439-443), The need for antibodies exhibiting increased affinity for the F protein is further increasing in order to provide potent MPV neutralization for the detection of MPV antigens (e.g. in diagnosis) and for the development of vaccines against MPV. exist.

In view of the above, an object of the present invention is to overcome the drawbacks of the prior art. In particular, it is an object of the present invention to provide antibodies that bind to the prefusion F protein of MPV with high binding affinity. It is also an object of the invention to provide antibodies that potently neutralize MPV infection.

This object is achieved by the subject matter described below and in the appended claims.

Although the invention is described in detail below, it is to be understood that the invention is not limited to the particular methodologies, protocols and reagents described herein, as these may vary. Additionally, it is to be understood that the terminology used herein is not intended to limit the scope of the invention, which is limited only by the claims appended hereto. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Elements of the invention will now be described. Although these elements are listed with particular embodiments, it is to be understood that they can be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the invention to only those explicitly described embodiments. This description is to be understood to support and encompass embodiments in combination with explicitly described embodiments having any number of disclosed elements. Furthermore, any permutations and combinations of all elements described in this application should be deemed to be disclosed by the description of this application, unless the context suggests otherwise.

Throughout this specification and the claims that follow, unless the context otherwise requires, the term "comprise" and variations such as "comprises" and "comprising" are used as specified. It is understood that the inclusion of a member, integer or step is not meant to imply the exclusion of any other unspecified member, integer or step. The term "consisting of" is a specific embodiment of the term "comprising" and excludes any other unlisted member, integer, or step. In the context of the present invention, the term "comprise" includes the term "consist of." Thus, the term "comprising" encompasses "including" and "consisting"; for example, a composition "comprising" X may consist only of X, or , for example, may include any additional configurations, such as X+Y.

The terms "a" and "an" and "The" and similar references used in the context of describing the invention (in particular in the context of the claims), and similar references, refer to or unless clearly contradicted by the context, should be construed to include both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand way of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The term "substantially" does not exclude "completely"; for example, a composition "substantially free" of Y may be completely free of Y. If desired, the word "substantially" may be excluded from the definition of the present invention.

The term "about" in relation to a numerical value x means x±10%, such as x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x± means 20%.

As used herein, the term "disease" means (medical condition) in that all words reflect an abnormal condition of the human or animal body, or one of its parts, that impairs its normal functioning. The terms "disorder" and "condition" (as in to have a decreased duration or quality of life.

As used herein, reference to "treatment" of a subject or patient is intended to include prophylaxis, prophylaxis, attenuation, amelioration and treatment. The terms "subject" or "patient" are used interchangeably herein to mean all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is human.

Doses are often expressed in relation to body weight. Therefore, doses expressed as [g, mg, or other units]/kg (or g, mg, etc.) are usually expressed as "per kg of body weight" even if the term "body weight" is not explicitly mentioned. or g, mg, etc.)” refers to [g, mg, or other units].

"Bind" and similar references generally mean "specifically bind" and do not include non-specific adhesion.

As used herein, the term "antibody" refers to whole antibodies, antibody fragments (such as antigen-binding fragments), human antibodies, chimeric antibodies, humanized antibodies, as long as the characteristic properties according to the invention are retained. It encompasses various forms of antibodies, including, but not limited to, recombinant antibodies, and genetically engineered antibodies (mutant or mutant antibodies). In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a monoclonal antibody. For example, the antibody is a human monoclonal antibody.

As noted above, the term "antibody" generally also includes antibody fragments. Fragments of antibodies may retain the antigen binding activity of the antibody. Such fragments are called "antigen-binding fragments." Antigen-binding fragments include, but are not limited to, single chain antibodies, Fabs, Fab', F(ab')2, Fv or scFv. Fragments of antibodies can be obtained from antibodies by methods involving digestion with enzymes such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of antibodies can be obtained by recombinant means, for example by cloning and expressing part (fragment) of the heavy and/or light chain sequences. The invention also encompasses single chain Fv fragments (scFv) derived from the heavy and light chains of the antibodies of the invention. For example, the invention includes scFvs that include CDRs derived from antibodies of the invention. Also included are heavy chain or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies, e.g., one in which the heavy and light chain variable domains are joined by a peptide linker. Chain Fv is also included. Antibody fragments of the invention may be contained in a variety of structures known to those skilled in the art. Furthermore, the sequences of the invention may be components of multispecific molecules in which the sequences of the invention target epitopes of the invention and other regions of the molecule bind other targets. Although this specification, including the claims, may in some places explicitly refer to antigen-binding fragments, antibody fragments, variants, and/or derivatives of antibodies, the term "antibody" It is understood to include all categories of antibodies, ie, antigen-binding fragments, antibody fragments, variants, and derivatives of antibodies.

Human antibodies are well known in the state of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies may also be produced in transgenic animals (eg, mice) that, upon immunization, are capable of producing a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies upon antigen challenge (e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 ( 1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The technique of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner , P., et al., J. Immunol. 147 (1991) 86-95). In some embodiments, human monoclonal antibodies are prepared using improved EBV-B cell immortalization. Improved EBV-B cell immunization has been linked to Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies. from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. As used herein, the term "variable region" (light chain variable region (V _L ), heavy chain variable region (V _H )) refers to the region directly involved in the binding of an antibody to an antigen. Each light chain and heavy chain pair is shown.

Antibodies of the invention can be of any isotype (eg, IgA, IgG, IgM, ie, alpha, gamma, or mu heavy chain). For example, the antibody is of the IgG type. Within the IgG isotype, antibodies may be of the IgG1, IgG2, IgG3 or IgG4 subclass, such as IgG1. Antibodies of the invention may have kappa or lambda light chains. In some embodiments, the antibody is of type IgG1 and has a kappa light chain.

Antibodies according to the invention may be provided in purified form. Typically, the antibody is present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition is other polypeptides, usually less than 60%, more usually less than 50%. It is composed of polypeptides.

Antibodies according to the invention may be immunogenic in human and/or non-human (or xenogeneic) hosts, such as mice. For example, an antibody may have an idiotope that is immunogenic in a non-human host, but not in a human host. Antibodies of the invention for human use include antibodies that cannot be easily isolated from a host such as a mouse, goat, rabbit, rat, non-primate mammal, and generally cannot be obtained from humanized or xenogeneic mice. This includes things that cannot be done.

As used herein, a "neutralizing antibody" is one that is capable of neutralizing the ability of a pathogen to initiate and/or perpetuate an infection in a host, i.e., prevent, inhibit, reduce, impede. ) or something that can interfere. The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies, alone or in combination, can be used as prophylactic or therapeutic agents in appropriate formulation, in conjunction with active vaccination, as diagnostic tools, or as production tools as described herein. can be done.

As used herein, the term "mutation" relates to a change in a nucleic acid and/or amino acid sequence compared to a reference sequence, eg, a corresponding genomic sequence. Mutations (e.g., when compared to a genomic sequence) can be, for example, somatic (naturally occurring) mutations, natural mutations, induced mutations, e.g. induced by enzymes, chemicals or radiation, or site-directed mutagenesis. (a molecular biological method for making specific and intentional changes in nucleic acid and/or amino acid sequences). Accordingly, the term "mutation" or "mutating" should be understood to also include physically creating a variation, for example in a nucleic acid or amino acid sequence. Mutations include substitutions, deletions and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. In order to effect mutations in the amino acid sequence, mutations may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutant polypeptide. Mutations can be made, for example, by changing the codons of a nucleic acid molecule encoding one amino acid (e.g., by site-directed mutagenesis), by introducing codons encoding different amino acids, or by synthesizing sequence variants (e.g., by site-directed mutagenesis). For example, by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and without having to mutate one or more nucleotides of the nucleic acid molecule, one can synthesize a nucleic acid molecule containing a nucleotide sequence encoding a variant of the polypeptide. design).

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. incorporated into the book. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

It is understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein, which may vary. Additionally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. I hope you understand that. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

(Antibodies and antigen-binding fragments thereof)
In a first aspect, the invention provides an antibody (isolated) that binds (specifically) to the F protein (fusion protein) of a metapneumovirus (MPV), in particular a human metapneumovirus (hMPV). Antigen-binding fragments are provided.

The MPV F protein is a type I transmembrane surface protein with an N-terminal truncated signal peptide and a membrane anchor near the C-terminus. The MPV F protein assembles into homotrimers and is synthesized as an inactive F0 precursor that is activated by cleavage. The F protein is formed by three domains (DI to DIII), a fusion peptide (FP) and three heptad repeat regions (HR-A, -B and -C). The MPV F glycoprotein induces viral penetration by fusion between the virion envelope and the plasma membrane of the host cell. The N-terminus of the F subunit is generated by proteolytic cleavage and contains the fusion peptide, which inserts directly into the target membrane to initiate fusion. After binding to the target cell and subsequent activation, the metastable prefusion F protein undergoes a series of structural rearrangements that result in the insertion of the fusion peptide into the target cell membrane, and subsequently as the viral and cell membranes line up. undergoes the formation of a stable helical bundle that forms. These structural changes result in the formation of a stable post-fusion F protein. After infection, the F protein expressed on the cell surface of infected cells can mediate fusion with adjacent uninfected cells forming large syncytia.

In some embodiments, the antibody or antigen-binding fragment thereof binds to the prefusion F protein of MPV, particularly human metapneumovirus (hMPV).

The prefusion F protein is the relevant conformation to block viral entry. Therefore, antibodies that recognize the prefusion conformation of the MPV F protein are particularly effective at neutralizing.

In some embodiments, the binding affinity of the antibody or antigen-binding fragment thereof is higher for the prefusion F protein than for the postfusion F protein. Thus, the antibody or antigen-binding fragment thereof may be selective for pre-fusion F protein (over post-fusion F protein). Recognition of the abundant post-fusion F protein, which may act as a decoy, consumes the antibody, thereby reducing its effectiveness, which is preferably reduced or avoided.

More specifically, a concentration of the antibody or its antigen that is at least 100 times higher for 50% antibody binding to the post-fusion F protein of the MPV than for 50% antibody binding to the pre-fusion F protein of the MPV. Binding fragments may be required.

Standard methods for assessing binding of antibodies or antigen-binding fragments thereof according to the invention are known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay). Thereby, the relative affinity of antibody binding can be determined by measuring the concentration of antibody required to achieve 50% maximum binding at saturation (EC ₅₀ ).

An exemplary standard ELISA may be performed as follows: the ELISA plate has enough of the protein/complex/particle (e.g., MPV F protein in prefusion or postfusion conformation) to be tested for antibody binding. (eg, 1 μg/ml). The plate may then be incubated with the antibody to be tested. After washing, antibody binding can be determined using a labeled antibody that recognizes the test antibody, eg, goat anti-human IgG conjugated to alkaline phosphatase. The plate may then be washed, the required substrate (eg, p-NPP) added, and the plate read at, eg, 405 nm. The relative affinity of antibody binding can be determined by measuring the concentration of mAb (EC ₅₀ ) required to achieve 50% maximum binding at saturation. EC ₅₀ values may be calculated by interpolation of the binding curve fitting a 4-parameter non-linear regression with variable slope.

In some embodiments, the antibody or antigen-binding fragment thereof neutralizes MPV, particularly hMPV infection. Antibodies and antigen-binding fragments of the invention may have high neutralizing capacity. The concentration of the antibody of the invention required for 50% neutralization of MPV is, for example, about 500 ng/ml or less. In certain embodiments, the concentration of antibodies of the invention required for 50% neutralization of MPV is about 500, 450, 400, 350, 300, 250, 200, 175, 150, 125 or about 100 ng/ml or less. . This means that only a low concentration of antibody is required for 50% neutralization of MPV. Specificity and potency can be measured using standard assays known to those skilled in the art.

Those skilled in the art are aware of various standard "neutralization assays" for studying and quantifying neutralization, for example, in the laboratory. For neutralization assays, the virus (to be neutralized) is typically grown in cells and/or cell lines. For example, in a neutralization assay, cultured cells may be incubated with a fixed amount of MPV (hMPV) in the presence (or absence) of the antibody being tested. For example, flow cytometry can be used as a readout. Alternatively, other lead-outs are also possible.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to F proteins of MPV subgroups A1, A2, B1, and B2, ie, all four subgroups of MPV. Thus, antibodies or antigen-binding fragments thereof may neutralize infection with MPV subgroups A1, A2, B1, and B2, ie, all four subgroups of MPV. The amino acid sequences of MPV G and F proteins are classified into groups A and B, and are further divided into four subgroups: A1, A2, B1 and B2. As shown in the accompanying examples, antibodies or antigen-binding fragments of the invention specifically bind to all four subgroups of MPV: A1, A2, B1, and B2. Furthermore, in some embodiments, antibodies or antigen-binding fragments of the invention potently neutralize all four subgroups of MPV: A1, A2, B1, and B2.

In some embodiments, the antibody or antigen-binding fragment thereof binds the same (or overlapping) epitope as MPE8 (e.g., Corti et al., 2013, Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501: 439-443 (which is incorporated herein by reference). MPE8 binds to an epitope near the central section of the RSV (and possibly MPV) F ectodomain at the intersection of the DI, DII, and DIII domains from the two subunits of the F trimer (also called "antigenic site III"). It is stated that

In other embodiments, the antibody or antigen-binding fragment thereof binds to an epitope on the MPV F protein that is different from (and non-overlapping with) the epitope of MPE8 (ie, different from antigenic site III).

Generally, an antibody or antigen-binding fragment thereof according to the invention may comprise (at least) three complementarity determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. Generally, complementarity determining regions (CDRs) are hypervariable regions present in heavy and light chain variable domains. Typically, the CDRs of an antibody's heavy chain and the linked light chain together form an antigen receptor. Typically, the three CDRs (CDR1, CDR2, and CDR3) are arranged non-contiguously in the variable domain. Antigen receptors are typically composed of two variable domains (on two different polypeptide chains, i.e., a heavy chain and a light chain: a heavy chain variable region (VH) and a light chain variable region (VL)). There are typically six CDRs for each antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). For example, a classical IgG antibody molecule typically has two antigen receptors and therefore contains 12 CDRs. The CDRs on the heavy and/or light chains may be separated by framework regions, whereby framework regions (FRs) are regions in variable domains that are less "variable" than the CDRs. For example, a variable region (or each variable region) may be composed of four framework regions separated by three CDRs.

The sequences of the heavy and light chains of an exemplary antibody of the invention were determined, including three different CDRs on the heavy chain and three different CDRs on the light chain. CDR amino acid positions are defined by the IMGT numbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012) be done.

In some embodiments, the antibody or antigen-binding fragment thereof has (i) a heavy chain CDR1, CDR2, having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and CDR3 sequences, and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7, respectively; or (ii) SEQ ID NO: 1, respectively; heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 2, and SEQ ID NO: 3, and with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity; or (iii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. chain CDR1, CDR2, and CDR3 sequences and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, respectively; or (iv) Heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. light chain CDR1, CDR2, and CDR3 sequences that have at least 70% sequence identity with the amino acid sequence of.

In some embodiments, the antibody or antigen-binding fragment thereof has (i) heavy chain CDR1, CDR2, having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and CDR3 sequences, and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7, respectively; or (ii) SEQ ID NO: 1, respectively; heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 2, and SEQ ID NO: 3, and with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. (iii) light chain CDR1, CDR2, and CDR3 sequences that have at least 80% sequence identity; or (iii) chain CDR1, CDR2, and CDR3 sequences and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, respectively; or (iv) Heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. light chain CDR1, CDR2, and CDR3 sequences that have at least 80% sequence identity with the amino acid sequence of.

As used throughout this specification, "sequence identity" is generally calculated with respect to the entire length of the reference sequence (ie, the sequence described in this application). Percentage identity as referred to herein refers to, for example, the National Center for Biotechnology Information (NCBI) [Blosum 62 matrix; gap open penalty =11 and gap extension penalty=1] by methods known in the art, such as blasting, using default parameters specified by [gap extension penalty=1].

A "sequence variant" has an altered sequence in which one or more amino acids in the reference sequence are deleted or substituted, and/or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the modification, an amino acid sequence variant has an amino acid sequence that is at least 70% identical to the reference sequence. A variant sequence that is at least 70% identical has no more than 30 modifications, ie, any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence. A "sequence variant" may maintain the functionality of the reference sequence (eg, binding to the F protein of MPV in this case).

In general, although it is possible to have non-conservative amino acid substitutions, substitutions are usually conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the reference sequence. . As examples, conservative amino acid substitutions include the replacement of one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine and isoleucine, with another; one hydroxyl-containing amino acid, such as serine and threonine, with another amino acid; substitution of one acidic residue, such as glutamic acid or aspartic acid, with another amino acid; substitution of one amide-containing residue, such as asparagine and glutamine, with another amino acid; one aromatic residue , the replacement of one basic residue, such as lysine, arginine and histidine, with another amino acid; and one small amino acid, such as alanine, serine, threonine, cysteine. and the replacement of glycine with another amino acid.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusions to the N-terminus or C-terminus of an amino acid sequence to a reporter molecule or enzyme.

Antibodies of the invention or antigen-binding fragments thereof (i) have at least 90% (e.g., 91%, 92%, 93%, 94%, 95%) the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; %, 96%, 97%, 98%, 99% or more) sequence identity, and the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. a light chain CDR1, CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with or (ii) at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%) of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; %, 98%, 99% or more) and at least 90% (e.g. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more); or (iii ) at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% (e.g., 91%, 92%, or more) sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, respectively. (iv) light chain CDR1, CDR2, and CDR3 sequences having sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, 99% or more; No. 13, and at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the amino acid sequence of SEQ ID No. 14 heavy chain CDR1, CDR2, and CDR3 sequences, and the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, at least 90% (e.g., 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more) sequence identity.

In some embodiments, the antibody or antigen-binding fragment thereof:
- a heavy chain CDR1 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1;
- a heavy chain CDR2 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2;
- a heavy chain CDR3 sequence having at least 85% or 90% sequence identity with the amino acid sequence of SEQ ID NO: 3;
- a light chain CDR1 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4;
- a light chain CDR2 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5 or 6; and - a light chain CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7.

More specifically, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 1;
- heavy chain CDR2 sequence according to SEQ ID NO: 2;
- a heavy chain CDR3 sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3;
- light chain CDR1 sequence according to SEQ ID NO: 4;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 5 or 6; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 7.

In some embodiments, the C-terminal Asp(D) residue of SEQ ID NO: 3 is substituted. More specifically, the C-terminal Asp residue of SEQ ID NO: 3 may be substituted with another polar amino acid. Examples of polar amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine and tyrosine. For example, the C-terminal aspartic acid residue of SEQ ID NO: 3 may be substituted with a histidine residue. Accordingly, the antibody or antigen-binding fragment thereof may comprise a heavy chain CDR3 sequence according to SEQ ID NO: 3 or 10.

Therefore, an antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 1;
- heavy chain CDR2 sequence according to SEQ ID NO: 2;
- heavy chain CDR3 sequence according to SEQ ID NO: 3;
- light chain CDR1 sequence according to SEQ ID NO: 4;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 5 or 6; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 7.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 1;
- heavy chain CDR2 sequence according to SEQ ID NO: 2;
- heavy chain CDR3 sequence according to SEQ ID NO: 10;
- light chain CDR1 sequence according to SEQ ID NO: 4;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 5 or 6; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 7.

In other embodiments, the antibody or antigen-binding fragment thereof:
- a heavy chain CDR1 sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 12;
- a heavy chain CDR2 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 13;
- a heavy chain CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14;
- a light chain CDR1 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 15;
- a light chain CDR2 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 18. You can stay there.

More specifically, the antibody or antigen-binding fragment thereof:
- a heavy chain CDR1 sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 12;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence according to SEQ ID NO: 18.

In some embodiments, one or more of the heavy chain variable region amino acid residues N34, S36, and C38 (corresponding to N6, S8, and C10 of SEQ ID NO: 12, respectively) are substituted. More specifically,
- N34 (corresponding to N6 in SEQ ID NO: 12) may be substituted with another polar amino acid such as Gln (Q) or Ser (S);
-S36 (corresponding to S8 in SEQ ID NO: 12) may be substituted with another small amino acid such as Ala (A); and/or -C38 (corresponding to C10 in SEQ ID NO: 12) may be substituted with Ser ( S), Ala (A) or Tyr (Y).

Examples of polar amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine and tyrosine. Examples of small amino acids include alanine, serine, threonine, glycine, cysteine, proline, asparagine and aspartic acid, with alanine, glycine and serine being particularly small.

The heavy chain CDR1 sequence, antibody or antigen binding fragment thereof, may differ by up to 3 amino acids from SEQ ID NO: 12, particularly as described above. In some embodiments, the heavy chain CDR1 sequence, antibody or antigen-binding fragment thereof, differs from SEQ ID NO: 12 by a single amino acid substitution, which may be selected from N34, S36, and C38, as described above. In other embodiments, the heavy chain CDR1 sequence, antibody or antigen-binding fragment thereof, differs from SEQ ID NO: 12 by (exactly) two amino acid substitutions, which are N34/S36, N34/C38 as described above. and S36/C38, for example, the amino acid residues of N34/C38 may be substituted as described above.

For example, the antibody or antigen-binding fragment thereof may include a heavy chain CDR1 sequence set forth in any one of SEQ ID NOs: 12, 21, 23, 25, 27, 29, 31, and 33.

Therefore, an antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 12;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 21;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 23;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 25;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 27;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 29;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence according to SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 31;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

Alternatively, the antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 33;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- a light chain CDR2 sequence as set forth in SEQ ID NO: 16 or 17; and - a light chain CDR3 sequence as set forth in SEQ ID NO: 18.

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%) of SEQ ID NO: 8; , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or more) and 70% or more (e.g., 71%) with SEQ ID NO:9. , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity Including area (VL). Thereby, the CDR sequences defined above (the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or 10, respectively; and SEQ ID NO: 4, SEQ ID NO: 5, or 6, respectively; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 7).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 70% or more (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%) of SEQ ID NO: 19; , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or more) and 70% or more (e.g., 71%, 72%) with SEQ ID NO:20. , 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). include. Thereby, the CDR sequences defined above (respectively SEQ ID NO: 12, 21, 23, 25, 27, 29, 31 or 33; SEQ ID NO: 13; and the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 20, respectively).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%) of SEQ ID NO: 8; , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more) and 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%) with SEQ ID NO: 9. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more). Thereby, the CDR sequences defined above (the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or 10, respectively; and SEQ ID NO: 4, SEQ ID NO: 5, or 6, respectively; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 7).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%) of SEQ ID NO: 19; , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more) and 75% or more (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%) with SEQ ID NO: 20. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more). Thereby, the CDR sequences defined above (respectively SEQ ID NO: 12, 21, 23, 25, 27, 29, 31 or 33; SEQ ID NO: 13; and the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 20, respectively).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%) of SEQ ID NO: 8; , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) chain variable region, and 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%) , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity. Thereby, the CDR sequences defined above (the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or 10, respectively; and SEQ ID NO: 4, SEQ ID NO: 5, or 6, respectively; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 7).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%) of SEQ ID NO: 19; , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) chain variable region, and 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%) with SEQ ID NO: , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity. Thereby, the CDR sequences defined above (respectively SEQ ID NO: 12, 21, 23, 25, 27, 29, 31 or 33; SEQ ID NO: 13; and the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 20, respectively).

In some embodiments, the antibody of the invention or antigen-binding fragment thereof has (i) 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%) of SEQ ID NO: 8; , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity a light chain variable region comprising an amino acid sequence. Thereby, the CDR sequences defined above (the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or 10, respectively; and SEQ ID NO: 4, SEQ ID NO: 5, or 6, respectively; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 7).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%) of SEQ ID NO: 19; , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and 85% or more (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity a light chain variable region comprising an amino acid sequence. Thereby, the CDR sequences defined above (respectively SEQ ID NO: 12, 21, 23, 25, 27, 29, 31 or 33; SEQ ID NO: 13; and the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 20, respectively).

In some embodiments, the antibodies of the invention or antigen-binding fragments thereof have (i) 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%) of SEQ ID NO: 8; , 98%, 99% or more) and 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity. Thereby, the CDR sequences defined above (the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or 10, respectively; and SEQ ID NO: 4, SEQ ID NO: 5, or 6, respectively; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 7).

In some embodiments, the antibody of the invention or antigen-binding fragment thereof has (i) 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%) of SEQ ID NO: 19; , 98%, 99% or more) and 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity. Thereby, the CDR sequences defined above (respectively SEQ ID NO: 12, 21, 23, 25, 27, 29, 31 or 33; SEQ ID NO: 13; and the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 20, respectively).

In some embodiments, an antibody of the invention or antigen-binding fragment thereof (i) has 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 8. a heavy chain variable region comprising an amino acid sequence; and a light chain variable region comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 9. . Thereby, the CDR sequences defined above (the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or 10, respectively; and SEQ ID NO: 4, SEQ ID NO: 5, or 6, respectively; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 7).

In some embodiments, an antibody of the invention or antigen-binding fragment thereof (i) has 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 19. a heavy chain variable region comprising an amino acid sequence; and a light chain variable region comprising an amino acid sequence having 95% or more (e.g., 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 20. include. Thereby, the CDR sequences defined above (respectively SEQ ID NO: 12, 21, 23, 25, 27, 29, 31 or 33; SEQ ID NO: 13; and the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 14; and the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16 or 17, and SEQ ID NO: 20, respectively).

More specifically, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9. .

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:22 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:26 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:30 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20.

Alternatively, the antibody or antigen-binding fragment thereof may include a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20.

Exemplary antibodies of the invention, namely antibodies MPF5_VH117D (MPF5) and MPF5_VH117H, MPE33, MPE33_S36A, MPE33_N34Q, MPE33_N34S, MPE33_C38S, MPE33_C38A, MPE33_C38Y and MPE33_N34S_ The CDR and VH/VL sequences of C38Y are shown in Table 1 below.

In some embodiments, antibodies of the invention are human antibodies. In some embodiments, antibodies of the invention are monoclonal antibodies. For example, antibodies of the invention may be human monoclonal antibodies.

Antibodies of the invention may be of any isotype (eg, IgA, IgG, IgM, ie, α, γ, or μ heavy chain). For example, the antibody may be of the IgG type. Within the IgG isotype, antibodies may be of the IgG1, IgG2, IgG3 or IgG4 subclass, eg IgG1. Antibodies of the invention may have kappa or lambda light chains. In some embodiments, the antibody is of type IgG1 and has a lambda or kappa light chain.

In some embodiments, the antibody is of the human IgG1 type. Antibodies may be of any allotype. The term "allotype" refers to allelic variation found among IgG subclasses. For example, the antibody may be of the G1m1 (or G1m(a)) allotype, the G1m2 (or G1m(x)) allotype, the G1m3 (or G1m(f)) allotype, and/or the G1m17 (or Gm(z)) allotype. Good too. The G1m3 and G1m17 allotypes are located at the same position in the CH1 domain (position 214 according to EU numbering). G1m3 corresponds to R214 (EU), while G1m17 corresponds to K214 (EU). The G1m1 allotype is located in the CH3 domain (positions 356 and 358 (EU)) and refers to the substitutions E356D and M358L. The G1m2 allotype refers to the substitution of alanine at position 431 (EU) with glycine. The G1m1 allotype can be combined with the G1m3 or G1m17 allotype, for example. In some embodiments, the antibody is of the allotype G1m3 without G1m1 (G1m3,-1). In some embodiments, the antibody is of the G1m17,1 allotype. In some embodiments, the antibody is of the G1m3,1 allotype. In some embodiments, the antibody is of the allotype G1m17 without G1m1 (G1m17,-1). Optionally, these allotypes may be combined (or not) with the G1m2, G1m27, or G1m28 allotypes. For example, the antibody may be of the G1m17,1,2 allotype.

In some embodiments, antibodies of the invention or antigen-binding fragments thereof include an Fc portion. The Fc portion may be derived from human origin, such as human IgG1, IgG2, IgG3, and/or IgG4, such as human IgG1.

As used herein, the term "Fc portion" refers to a sequence derived from a portion of an immunoglobulin heavy chain. The immunoglobulin heavy chain begins at the hinge region just upstream of the papain cleavage site (e.g., residue 216 of native IgG, the first residue of the heavy chain constant region is 114), and begins at the C of the immunoglobulin heavy chain. ends at the end. Thus, the Fc portion may be a complete Fc portion or a portion thereof (eg, a domain). A complete Fc portion includes at least a hinge domain, a CH2 domain, and a CH3 domain (eg, EU amino acid positions 216-446). An additional lysine residue (K) may be present at the extreme C-terminus of the Fc portion, but is often cleaved from the mature antibody.

Each of the amino acid positions within the Fc portion is numbered according to the art-recognized Kabat EU numbering system (e.g., Kabat et al., in “Sequences of Proteins of Immunological Interest”, US Dept. Health and Human Services, 1983). and 1987, so please refer to them). EU index or EU index, such as Kabat, or EU numbering refers to the numbering of EU antibodies (Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ. The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci US A. 1969;63(1):78-85; Kabat EA, National Institutes of Health (US) Office of the Director, “Sequences of Proteins of Immunological Interest”, 5th edition, Bethesda, MD: US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 199, incorporated herein by reference in its entirety.)
In some embodiments, in the context of the present invention, an Fc portion comprises a hinge (e.g., upper, middle, and/or lower hinge region) domain, CH2 domain, CH3 domain, or a variant, portion, or fragment thereof. including at least one of them. The Fc portion may include at least a hinge domain, a CH2 domain, or a CH3 domain. The Fc portion may be a complete Fc portion. The Fc portion may also contain one or more amino acid insertions, deletions, or substitutions as compared to the naturally occurring Fc portion. For example, at least one of the hinge domain, CH2 domain, or CH3 domain (or portion thereof) may be deleted. For example, the Fc portion may include (i) a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof); (iv) a hinge domain (or a portion thereof); (v) a CH2 domain (or a portion thereof); or (vi) a CH3 domain. , or portions thereof.

The Fc portion may be modified to vary in amino acid sequence from the complete Fc portion of a naturally occurring immunoglobulin molecule while retaining at least one desirable function conferred by the naturally occurring Fc portion; will be understood by those skilled in the art. Such functions include Fc receptor (FcR) binding, antibody half-life regulation, ADCC function, protein A binding, protein G binding, and complement fixation. The portions of the naturally occurring Fc portion that are involved in and/or essential for such functions are well known to those skilled in the art.

For example, to activate complement cascade C1q binding to at least two molecules of IgG1 or one molecule of IgM attached to an antigen target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R. (Mol. Immunol. 22 (1985) 161-206) describes that the heavy chain region containing amino acid residues 318 to 337 is involved in complement fixation. Using site-directed mutagenesis, Duncan, A. R., and Winter, G. (Nature 332 (1988) 738-740) reported that Glu318, Lys320 and Lys322 form the binding site for C1q. The role of Glu318, Lys320 and Lys322 residues in C1q binding was confirmed by the ability of short synthetic peptides containing these residues to inhibit complement-mediated lysis.

For example, FcR binding can be mediated by the interaction of the Fc portion (of the antibody) with Fc receptors (FcR), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and are responsible for the removal of antibody-coated pathogens by phagocytosis of immune complexes, as well as for antibody-coated pathogens via antibody-dependent cell-mediated cytotoxicity. (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are called FcγRs, those for IgE are called FcεR, those for IgA are FcαR, etc., and the neonatal Fc receptors are called FcRn. Called. Fc receptor binding is described, for example, in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas , M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcγR) contributes to a wide variety of effector functions, including phagocytosis, antibody-dependent cytotoxicity, and release of inflammatory mediators, as well as regulation of immune complex clearance and antibody production. induce. Thus, the Fc portion may provide cross-linking of the receptor (FcγR). In humans, three classes of FcγR have been characterized: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) complex FcγRIIA, which binds with low affinity to FcγRIIA, is widely expressed, but is particularly expressed on leukocytes, which are known to play a central role in antibody-mediated immunity. It is known to be divided into FcγRIIB and FcγRIIC, which have different functions in the immune system, but bind with low affinity similar to IgG-Fc, and the ectodomains of these receptors are highly homologous. (iii) FcγRII (CD32), which binds to IgG with intermediate to low affinity and has two types (NK cells, macrophages, eosinophils and some monocytes, and FcγRIII (CD16), which is found on T cells and exists as FcγRIIA, which mediates ADCC, and FcγRIIB, which is highly expressed on neutrophils. FcγRIIA is found in many cells involved in killing (eg, macrophages, monocytes, neutrophils) and appears capable of activating the killing process. FcγRIIB appears to play a role in the inhibitory process and is found on B cells, macrophages, and mast cells and eosinophils. Importantly, 75% of all FcγRIIB is found in the liver (Ganesan, L. P. et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988). FcγRIIB is abundantly expressed on the Liver Sinusoidal Endothelium called LSEC, and Kupffer cells in the liver and LSEC are the major sites of small immune complex clearance (Ganesan, L. P. et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

Accordingly, the antibodies of the invention and antigen-binding fragments thereof may bind to the Fc region of FcγRIIb, e.g., antibodies that contain an Fc portion for binding to FcγRIIb, particularly antibodies of the IgG type, e.g., IgG type antibodies. Furthermore, as described in Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcγRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933. , S267E and L328F mutations, the Fc portion can be designed to enhance FcγRIIB binding. Thereby, the clearance of immune complexes can be enhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). Therefore, the antibodies of the present invention or antigen-binding fragments thereof can be used in combination with Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcγRIIb with Fc-engineered antibodies. Molecular Immunology 45 , 3926-3933, may include engineered Fc portions with the S267E and L328F mutations.

On B cells, it appears to function by inhibiting further immunoglobulin production and isotype switching, such as the IgE class. In macrophages, FcγRIIB acts to inhibit phagocytosis mediated through FcγRIIA. On eosinophils and mast cells, the b form may serve to suppress activation of these cells through IgE binding to its distinct receptors.

With respect to FcγRI binding, modification in at least one of the natural IgGs of E233-G236, P238, D265, N297, A327 and P329 reduces binding to FcγRI. IgG2 residues at positions 233-236, substituted with IgG1 and IgG4, reduced binding to FcγRI by 10 ³ -fold and eliminated human monocyte responses to antibody-sensitized erythrocytes (Armour, KL, et al. J. Immunol. 29 (1999) 2613-2624). Regarding FcγRII binding, decreased binding to FcγRIIA is seen for at least one IgG mutation, eg, E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414. With respect to FcγRIII binding, decreased binding to FcγRIIIA is, for example, E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376. Found for at least one mutation. Mapping of binding sites on human IgG1 for Fc receptors, mutation sites and methods described above for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001). 6591-6604. For example, single (S239D or I332E), double (S239D/I332E), and triple mutations (S239D/I332E/A330L) improved affinity for human FcγRIIIa. Furthermore, addition of the G236A mutation to S239D/I332E not only improved the FcγRIIa:FcγRIIb ratio but also enhanced binding to FcγRIIIa. Therefore, the G236A/S239D/A330L/I332E mutations were described to enhance FcγRIIa and FcγRIIIa engagement.

Two regions of native IgG Fc are important for binding to FcγRII, namely: (i) the lower hinge region of IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering); ii) adjacent regions of the CH2 domain of IgG Fc, especially the loops and strands of the upper CH2 domain adjacent to the lower hinge region, e.g. the region of P331 (Wines, B.D., et al., J. Immunol. 2000; 164: 5313 - 5318 ) appears to be important for the interaction of FcγRII and IgG. Furthermore, FcγRI appears to bind to the same site on IgG Fc, whereas FcRn and protein A bind to different sites on IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D., et al. al., J. Immunol. 2000; 164: 5313 - 5318).

For example, the Fc portion may include or consist of at least a portion of an Fc portion known in the art to be required for FcRn binding or extended half-life. good. Alternatively, or in addition, the Fc portion of an antibody of the invention comprises at least a portion known in the art to be required for protein A binding and/or The Fc portion includes at least a portion of an Fc molecule known in the art that is required for protein G binding. The Fc portion may include at least a portion known in the art to be necessary for FcγR binding. Thus, as outlined above, the Fc portion includes (i) the lower hinge region of the native IgG Fc, specifically the amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the native The flanking regions of the CH2 domain of an IgG Fc, in particular the loops and strands in the upper CH2 domain adjacent to the lower hinge region (e.g. in the region of P331), e.g. A region of at least 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids of the upper CH2 domain between amino acids 320 and 340 of Fc (EU numbering) It may contain at least.

In some embodiments, antibodies of the invention or antigen-binding fragments thereof include an Fc region. As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by two or more Fc portions of an antibody heavy chain. For example, the Fc region may be a monomeric Fc region or a "single chain" Fc region (ie, a scFc region). A single-chain Fc region is composed of Fc portions linked into a single polypeptide chain (eg, encoded by a single contiguous nucleic acid sequence). Exemplary scFc regions are disclosed in WO2008/143954A2. The Fc region may be a dimer. "Dimeric Fc region" or "dcFc" refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. A dimeric Fc region may be a homodimer of two identical Fc portions (eg, of a naturally occurring immunoglobulin Fc region) or a heterodimer of two non-identical Fc portions.

The Fc portions of the Fc region may be of the same or different classes and/or subclasses. For example, the Fc portion may be derived from an immunoglobulin of the IgG1, IgG2, IgG3, or IgG4 subclass (eg, a human immunoglobulin). The Fc portions of the Fc region may be of the same class and subclass. However, the Fc region (or one or more Fc portions of an Fc region) may be chimeric, and a chimeric Fc region may include Fc portions from different immunoglobulin classes and/or subclasses. For example, the at least two Fc portions of a dimeric or single chain Fc region may be derived from different immunoglobulin classes and/or subclasses. Alternatively, or in addition, a chimeric Fc region may include one or more chimeric Fc moieties. For example, a chimeric Fc region or portion may include one or more portions derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), with the remainder of the Fc region or portion being different. It is of a subclass. For example, the Fc region or portion of an Fc polypeptide may include a CH2 and/or CH3 domain derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG4 subclass) and a CH2 and/or CH3 domain derived from an immunoglobulin of a second subclass (e.g., IgG3 subclass). ) may also contain a hinge region derived from an immunoglobulin. For example, the Fc region or portion may include a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). It may also contain a CH3 domain derived from. For example, a chimeric Fc region includes an Fc portion (e.g., a complete Fc portion) derived from an immunoglobulin of a first subclass (e.g., the IgG4 subclass) and an Fc portion (e.g., a complete Fc portion) from an immunoglobulin of a second subclass (e.g., the IgG1, IgG2, or IgG3 subclass). The Fc portion derived from immunoglobulin may also be included. For example, the Fc region or portion may include a CH2 domain derived from an IgG4 immunoglobulin and a CH3 domain derived from an IgG1 immunoglobulin. For example, the Fc region or portion may include a CH1 domain and a CH2 domain derived from an IgG4 molecule and a CH3 domain derived from an IgG1 molecule. For example, the Fc region or portion may include a portion of the CH2 domain from a particular subclass of antibodies, eg, CH2 domain EU positions 292-340. For example, the Fc region or portion may include amino acids 292-340 of CH2, which is derived from the IgG4 portion, and the remainder of CH2, which may be derived from the IgG1 portion (alternatively, amino acids 292-340 of CH2, which may be derived from the IgG1 portion, and IgG4 (residue of CH2 which may be derived from

Furthermore, the Fc region or portion may (alternatively or additionally) include, for example, a chimeric hinge region. For example, a chimeric hinge may be derived, for example, partially from IgG1, IgG2, or IgG4 molecules (e.g., upper and lower middle hinge sequences), and partially from IgG3 molecules (e.g., middle hinge sequences). You may. In another example, the Fc region or portion may include a chimeric hinge partially derived from IgG1 molecules and partially derived from IgG4 molecules. In another example, a chimeric hinge may include upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge may be created, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of the IgG4 hinge region. In other embodiments, the chimeric hinge can include amino acids at EU positions 233-236, where the amino acids are derived from an IgG2 antibody and/or are Ser228Pro mutated, and the remaining amino acids of the hinge are derived from an IgG4 antibody (e.g. , a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP). Further chimeric hinges that may be used in the Fc part of antibodies according to the invention are described in US2005/0163783A1.

In some embodiments, the Fc portion or Fc region comprises an amino acid sequence derived from a human immunoglobulin sequence (e.g., derived from an Fc region or Fc portion derived from a human IgG molecule), or configured. However, the polypeptide may contain one or more amino acids from another mammalian species. For example, a primate Fc portion or a primate binding site may be included in a subject polypeptide. Alternatively, one or more murine amino acids may be present in the Fc portion or region.

In some embodiments, the antibodies according to the invention, in particular in addition to the above-mentioned Fc part, comprise other parts derived from a constant region, in particular an IgG constant region, for example a (human) IgG1 constant region. The antibodies according to the invention may, in particular in addition to the above-mentioned Fc part, contain all other parts of the constant region, in particular all other parts of the constant region of IgG (for example (human) IgG1). .

Examples of constant region sequences are the amino acid sequences according to SEQ ID NOs: 35-37. For example, the amino acid sequence of IgG1 CH1-CH2-CH3 may be SEQ ID NO: 35 or a sequence variant thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9). , including 10 or more mutations), have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

As outlined above, antibodies according to the invention may contain a (complete) Fc region derived from human IgG1. In some embodiments, the antibody according to the invention comprises an IgG constant region, in particular a (complete) Fc region derived from human IgG1, as well as all other parts of the (human) IgG1 constant region. Also includes all other parts.

In some embodiments, an antibody according to the invention comprises a (complete) Fc part/Fc region, and interaction/binding with FcR is not impaired. In general, binding of antibodies to Fc receptors can be determined by various methods known to those skilled in the art, such as ELISA (Hessell AJ, Hangartner L, Hunter M, Havenith CEG, Beurskens FJ, Bakker JM, Lanigan CMS, Landucci G, Forthal DN, Parren PWHI, et al.: Fc receptor but not complement binding is important in antibody protection against HIV. Nature 2007, 449:101-104; Grevys A, Bern M, Foss S, Bratlie DB, Moen A, Gunnarsen KS, Aase A , Michaelsen TE, Sandlie I, Andersen JT: Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions. 2015, 194:5497-5508) or flow cytometry (Perez LG, Costa MR, Todd CA, Haynes BF, Montefiori DC: Utilization of immunoglobulin G Fc receptors by human immunodeficiency virus type 1: a specific role for antibodies against the membrane-proximal external region of gp41. J Virol 2009, 83:7397-7410; Piccoli L, Campo I, Fregni CS, Rodriguez BMF, Minola A, Sallusto F, Luisetti M, Corti D, Lanzavecchia A: Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis. Nat Commun 2015, 6:1-9) good.

Generally, antibodies according to the invention may be glycosylated. N-linked glycans attached to the CH2 domain of the heavy chain can, for example, affect the binding of C1q and FcR to glycosylated antibodies, which have lower affinity for these receptors. Thus, the CH2 domain of the Fc part of an antibody according to the invention may contain one or more mutations in which glycosylated residues are replaced by non-glycosylated residues. For example, the glycans of antibodies do not produce a human immunogenic response after administration.

Furthermore, antibodies according to the invention introduce (random) amino acid mutations in specific regions of the CH2 or CH3 domains of the heavy chain to improve their binding affinity for FcRs compared to unmodified antibodies. or can be modified by changing their serum half-life. Examples of such modifications include, but are not limited to, substitution of at least one amino acid from the heavy chain constant region selected from the group consisting of amino acid residues 250, 314, and 428. Further examples of such Fc modifications are described in Saxena A, Wu D. Advances in Therapeutic Fc Engineering - Modulation of IgG-Associated Effector Functions and Serum Half-life. Front Immunol. 2016;7:580, which is incorporated herein by reference. In some embodiments, the antibody may include a "YTE" mutation (M252Y/S254T/T256E; EU numbering). In some embodiments, the antibody may include mutations M428L and/or N434S in the heavy chain constant region (EU numbering).

Antibodies of the invention also include hybrid antibody molecules comprising six CDRs from an antibody of the invention as defined above and one or more CDRs from another antibody directed against the antigen. For example, antibodies may be bispecific. A bispecific (or multispecific) antibody or antigen-binding fragment thereof according to the invention may contain at least one specificity (antigen-binding site of the antibody) as described herein. In some embodiments, the bispecific (or multispecific) antibody or antigen-binding fragment thereof binds to two different epitopes of the MPV F protein. For this purpose, the specificities of two different antibodies of the invention may be combined, for example as described below for combinations of antibodies (but instead of a "cocktail", bispecific- or multispecific - the antibody may have a combination of specificities).

Variant antibodies are also included within the scope of the invention. Variants of the sequences listed in this application are therefore also included within the scope of the invention. Such variants include natural variants generated by somatic mutation in vivo during an immune response or in vitro during culture of immortalized B cell clones. Alternatively, variants may arise due to degeneracy in the genetic code, or may be produced due to errors in transcription or translation.

Antibodies of the invention may be provided in purified form. Typically, the antibody is present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition, usually less than 60%, more usually less than 50%. Consists of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (or xenogeneic) hosts, such as mice. In particular, an antibody may have an idiotope that is immunogenic in a non-human host, but not in a human host. In particular, antibodies of the invention for use in humans cannot be easily isolated from a host such as, for example, a mouse, goat, rabbit, rat, non-primate mammal, and are generally prepared by humanization or Including things that cannot be obtained from Xenomouse.

(nucleic acid)
In another aspect, the invention also provides a nucleic acid molecule comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof according to the invention as described above.

In some embodiments, the nucleic acid molecule is one or more polynucleotides described herein encoding an exemplary antibody of the invention (e.g., as described in Table 1 above), or Sequence variants (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 as described above) %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity).

Exemplary nucleic acid sequences encoding the CDR and VH/VL sequences of exemplary antibodies of the invention are shown in Table 2 below.

For example, the nucleic acid molecule may include (i) or (ii):
(i) a polynucleotide according to any one of SEQ ID NO: 45 or 54; and a polynucleotide according to any one of SEQ ID NO: 46 or 55;
(ii) Polynucleotide as described in any one of SEQ ID NO: 38 or 47; Polynucleotide as described in any one of SEQ ID NO: 39 or 48; Polynucleotide as described in any one of SEQ ID NO: 40 or 49 ; a polynucleotide according to any one of SEQ ID NO: 41 or 50; a polynucleotide according to any one of SEQ ID NO: 42, 43, 51, or 52; and any one of SEQ ID NO: 44 or 53 The described polynucleotide.

Examples of nucleic acid molecules and/or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules (such as rRNA, mRNA, miRNA, siRNA, or tRNA), or DNA molecules (such as cDNA). . The nucleic acid may encode the light chain and/or heavy chain of the antibody. In other words, the light and heavy chains of an antibody may be encoded by the same nucleic acid molecule (eg, in a bicistronic manner). Alternatively, the light and heavy chains of an antibody may be encoded by different nucleic acid molecules.

Because of the redundancy of the genetic code, the invention also includes sequence variants of nucleic acid sequences encoding the same amino acid sequence. A polynucleotide encoding an antibody (or complete nucleic acid molecule) may be optimized for expression of the antibody. For example, codon optimization of nucleotide sequences may be used to improve the efficiency of translation in expression systems for the production of antibodies. Additionally, the nucleic acid molecule may contain heterologous (i.e., non-naturally occurring) components that are not present on the same nucleic acid molecule as the coding sequence for (the heavy or light chain of) the antibody. For example, the nucleic acid molecule may contain May include heterologous promoters, heterologous enhancers, heterologous UTRs (eg, for optimal translation/expression), heterologous poly-A-tails, and the like.

A nucleic acid molecule is a molecule that includes a nucleic acid component. The term nucleic acid molecule typically refers to DNA or RNA molecules. It may be used synonymously with the term "polynucleotide," ie, a nucleic acid molecule may consist of a polynucleotide encoding an antibody. Alternatively, the nucleic acid molecule may also contain additional elements in addition to the polynucleotide encoding the antibody. Typically, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules such as base-modified DNA or RNA molecules, sugar-modified DNA or RNA molecules, or backbone-modified DNA or RNA molecules.

Generally, nucleic acid molecules may be manipulated to insert, delete, or alter specific nucleic acid sequences. Modifications from such manipulations include modifications to introduce restriction sites, modifications to modify codon usage, modifications to add or optimize transcriptional and/or translational regulatory sequences, etc. However, it is not limited to these. It is also possible to modify the nucleic acid to change the encoded amino acid. For example, introducing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions, and/or insertions into the amino acid sequence of the antibody. may be useful. Such point mutations can alter effector function, antigen binding affinity, post-translational modifications, immunogenicity, etc., and can introduce amino acids for the addition of covalent groups (e.g., labels). or tags (eg, for purification purposes) can be introduced. Alternatively, mutations in the nucleic acid sequence may be "silent", ie, not reflected in the amino acid sequence due to redundancy in the genetic code. Generally, mutations can be introduced at specific sites or randomly followed by selection (eg, molecular evolution). For example, one or more nucleic acids encoding either the light chain or the heavy chain of an (exemplary) antibody can be mutated randomly or specifically to introduce different properties to the encoded amino acids. can. Such changes may be the result of an iterative process in which the original change is retained and new changes at other nucleotide positions are introduced. Additionally, changes accomplished in independent steps may be combined.

In some embodiments, the polynucleotide (or (complete) nucleic acid molecule) encoding the antibody or antigen-binding fragment thereof may be codon-optimized. Those skilled in the art are aware of various tools for codon optimization, such as those described in: Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): or in: Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W526－31; or, for example, Genscript's OptimumGeneTM algorithm (described in US2011/0081708A1)
For example, a nucleic acid molecule of the invention comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 38-55; , at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The invention also provides a combination of a first and a second nucleic acid molecule, the first nucleic acid molecule comprising a polynucleotide encoding a heavy chain of an antibody of the invention or an antigen-binding fragment thereof; The molecule includes a polynucleotide encoding the corresponding light chain of the same antibody or the same antigen-binding fragment thereof. The above description of the (general) characteristics of the nucleic acid molecules of the invention applies accordingly to the first and second nucleic acid molecules of the combination. Accordingly, one or both of the polynucleotides encoding the antibody heavy and/or light chains or antigen-binding fragments thereof may be codon-optimized. For example, the combination comprises a nucleic acid sequence according to any one of SEQ ID NOs: 38-55; , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

The invention also provides a combination of first and second nucleic acid molecules,
- the first nucleic acid molecule comprises a polynucleotide encoding an antibody heavy chain or antigen-binding fragment thereof, the polynucleotide comprising (a) the nucleotide sequence set forth in SEQ ID NO: 38, 39 and 40; or (b) SEQ ID NO: 47, 48 and 49; and - the second nucleic acid molecule comprises a polynucleotide encoding an antibody light chain or antigen-binding fragment thereof, the polynucleotide comprising: (c) SEQ ID NOs: 41, 42; (or 43) and 44; or (d) the nucleotide sequences set forth in SEQ ID NOs: 50, 51 (or 52) and 53.

Such combinations typically encode antibodies of the invention or antigen-binding fragments thereof as described above. In this case too, the above explanations regarding the (general) characteristics of the nucleic acid molecules of the invention apply depending on the first and second nucleic acid molecules of the combination.

(vector)
Further included within the scope of the invention are vectors, such as expression vectors, comprising a nucleic acid molecule according to the invention. Vectors typically include the nucleic acid molecules described above.

The invention also provides that the first vector comprises a first nucleic acid molecule (for a combination of nucleic acid molecules) as described above, and the second vector comprises a second nucleic acid molecule (for a combination of nucleic acid molecules) as described above. A combination of first and second vectors is provided.

Vectors are typically recombinant, ie, non-naturally occurring, nucleic acid molecules. Thus, a vector may contain heterologous elements (ie, sequence elements of different origin in nature). For example, the vector may contain a multiple cloning site, a heterologous promoter, a heterologous enhancer, a heterologous selection marker (to identify cells containing the vector as compared to cells not containing the vector), and the like. Vectors in the context of the present invention are suitable for incorporating or carrying desired nucleic acid sequences. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors, and the like. A storage vector is a vector that allows convenient storage of nucleic acid molecules. Thus, the vector may, for example, contain a sequence corresponding to (the heavy chain and/or light chain of) the desired antibody according to the invention. Expression vectors may be used for the production of expression products such as RNA, eg, mRNA, or peptides, polypeptides, or proteins. For example, an expression vector may contain sequences necessary for transcription of a stretch of sequence of the vector, such as a (heterologous) promoter sequence. A cloning vector is a vector that typically contains a cloning site that can be used to incorporate nucleic acid sequences into the vector. The cloning vector may be, for example, a plasmid vector or a bacteriophage vector. A transcription vector may be a vector suitable for transcribing a nucleic acid molecule into a cell or organism, such as a viral vector. A vector in the context of the invention may be, for example, an RNA vector or a DNA vector. For example, a vector within the meaning of the present application comprises a cloning site, a selection marker such as an antibiotic resistance factor, and sequences suitable for propagation of the vector, such as an origin of replication. A vector in the context of this application may be a plasmid vector.

(cell)
In a further aspect, the invention also provides a cell expressing an antibody according to the invention or an antigen-binding fragment thereof; and/or a cell comprising a vector (or combination of vectors) according to the invention.

Examples of such cells include, but are not limited to, eukaryotic cells such as yeast cells, animal cells, or plant cells. Other examples of such cells include, but are not limited to, prokaryotic cells such as E. coli. In some embodiments, the cell is a mammalian cell, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER. C6 cells, NSO cells, human hepatocytes, myeloma cells, or hybridoma cells.

Cells may be transfected with vectors according to the invention, eg expression vectors. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (eg, mRNA) molecule, into a cell, eg, a eukaryotic or prokaryotic cell. In the context of the present invention, the term "transfection" encompasses any method known to those skilled in the art for introducing nucleic acid molecules into cells, such as mammalian cells. Such methods include, for example, electroporation, lipofection (e.g. based on cationic lipids and/or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymers (e.g. DEAE-dextran or polyethyleneimine, etc.) based transfections. In some embodiments, the introduction is non-viral.

Furthermore, the cells of the invention may be stably or transiently transfected with the vectors of the invention, for example in order to express the antibodies of the invention. In some embodiments, cells are stably transfected with a vector of the invention encoding an antibody of the invention. In other embodiments, cells are transiently transfected with a vector according to the invention encoding an antibody according to the invention.

Accordingly, the invention also provides recombinant host cells that heterologously express antibodies of the invention or antigen-binding fragments thereof. For example, the cells may be of another species other than antibodies (eg, CHO cells expressing human antibodies). In some embodiments, the cell type does not naturally express the antibody. Additionally, host cells may confer post-translational modifications (PTMs; eg, glycosylation) on antibodies that are not present in their native state. Such PTMs may result in functional differences (eg, reduced immunogenicity). Accordingly, the antibodies of the invention or antigen-binding fragments thereof may have different post-translational modifications than naturally produced antibodies (eg, antibodies of the immune response in humans).

(Production of antibodies)
Antibodies according to the invention can be produced by any method known in the art. For example, the general methodology for producing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983). In some embodiments, an alternative EBV immortalization method described in WO2004/076677 is used.

In some embodiments, the method described in WO2004/076677, which is incorporated herein by reference, is used. In this method, B cells producing antibodies of the invention are transformed with EBV and a polyclonal B cell activator. Additional stimulators of cell proliferation and differentiation may optionally be added during the transformation step to further increase efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not required. Immortalized B cells produced using these methods can then be cultured using methods known in the art and antibodies isolated therefrom.

Another exemplary method is described in WO2010/046775. In this method, plasma cells are cultured in limited numbers or as single plasma cells in microwell culture plates. Antibodies can be isolated from plasma cell cultures. Additionally, from plasma cell cultures, RNA can be extracted and PCR performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, cloned into an expression vector, and then transfected into HEK293T cells or other host cells. Cloning of nucleic acids in expression vectors, transfection of host cells, culturing of transfected host cells, and isolation of produced antibodies can be performed using any method known to those skilled in the art.

If desired, antibodies can be further purified using filtration, centrifugation, and various chromatographic methods (such as HPLC or affinity chromatography). Techniques for the purification of antibodies, such as monoclonal antibodies, including techniques for producing pharmaceutical grade antibodies, are well known in the art.

Standard techniques of molecular biology may be used to prepare DNA sequences encoding antibodies of the invention. The desired DNA sequence may be completely or partially synthesized using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

Any suitable host cell/vector system may be used for expression of DNA sequences encoding antibody molecules of the invention. Eukaryotic, eg, mammalian, host cell expression systems may be used for the production of antibody molecules, eg, complete antibody molecules. Suitable mammalian host cells include CHO cells, HEK293T cells, PER. Examples include, but are not limited to, C6 cells, NSO cells, myeloma cells, or hybridoma cells. Prokaryotic, eg, bacterial, host cell expression systems may also be used for the production of antibody molecules, eg, complete antibody molecules. Suitable bacterial host cells include, but are not limited to, E. coli cells.

The invention also provides the steps of culturing a (heterologous) host cell containing a vector encoding a nucleic acid of the invention under conditions suitable for expression of a protein from DNA encoding an antibody molecule of the invention; Provided is a method for producing an antibody molecule according to the present invention, comprising a step of isolating the antibody molecule.

For production of antibodies containing both heavy and light chains, cell lines are transfected with two vectors, a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide. may be done. Alternatively, a single vector can be used, the vector containing sequences encoding the light and heavy chain polypeptides.

An antibody according to the invention can be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, for example by using a vector according to the invention, and (ii) isolating the expressed antibody product. It may be produced by a process. Additionally, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for those producing antibodies of desired specificity or function.

The screening step can be performed by any immunoassay, such as ELISA, by staining of tissues or cells (including transfected cells), by neutralization assays, or by any method known in the art to identify the desired specificity or function. may be performed in one of many other ways. Assays may be selected on the basis of simple recognition of one or more antigens, or on the basis of additional desired functionality, e.g., selecting neutralizing antibodies rather than simply antigen-binding antibodies, to improve the properties of the target cells. (e.g. their signal transduction cascades, their shape, their rate of proliferation, their ability to influence other cells, their response to influences by other cells or by other reagents or by changing conditions, their One may choose to select antibodies that are capable of altering the differentiation state (e.g., the differentiation state of the antibody).

Individual transformed B cell clones may then be produced from positive transformed B cell cultures. The cloning step to isolate individual clones from a mixture of positive cells may be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or other methods known in the art. .

Nucleic acids from cultured plasma cells can be isolated, cloned, and expressed in HEK293T cells or other known host cells using methods known in the art.

Immortalized B cell clones or transfected host cells of the invention can be used in a variety of ways, including as a source of monoclonal antibodies, as a source of nucleic acids (DNA or mRNA) encoding monoclonal antibodies of interest, for research purposes, etc. It can be used for etc.

The invention also provides compositions comprising immortalized B memory cells or transfected host cells producing antibodies according to the invention.

Immortalized B cell clones or cultured plasma cells of the invention may also be used as a source of nucleic acids for cloning of antibody genes for subsequent recombinant expression. Expression from recombinant sources may be more common for pharmaceutical purposes than expression from B cells or hybridomas, for example because of stability, reproducibility, ease of culturing, etc.

Accordingly, the present invention also provides the steps of: (i) obtaining one or more nucleic acids (e.g., heavy chain and/or light chain mRNA) from a B cell clone or cultured plasma cell encoding an antibody of interest; ii) inserting a nucleic acid into an expression vector; and (iii) transfecting the vector into a (heterologous) host cell to enable expression of the antibody of interest in that host cell. Provided is a method for preparing.

Similarly, the present invention also provides the steps of: (i) sequencing nucleic acids from a B cell clone or cultured plasma cells encoding the antibody of interest; (ii) using the sequence information from step (i); , preparing a nucleic acid for insertion into a host cell to enable expression of an antibody of interest in that host cell. The nucleic acid is manipulated between steps (i) and (ii) to introduce restriction sites, alter codon usage, and/or optimize transcriptional and/or translational control sequences. Yes, but not necessarily.

Additionally, the invention also includes the step of transfecting a host cell with one or more nucleic acids encoding an antibody of interest, wherein the nucleic acids are derived from an immortalized B cell clone or cultured plasma cell of the invention. A method of preparing a transfected host cell is provided. Thus, the procedure for first preparing a nucleic acid and then using it to transfect a host cell can be performed by different people in different locations (eg, in different countries) at different times.

These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expressing antibodies for large scale pharmaceutical manufacturing. They can also be used as active ingredients in pharmaceutical compositions. Any suitable culture technique including, but not limited to, static culture, roller bottle culture, ascites fluid, hollow fiber bioreactor cartridges, modular mini-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, etc. ) can be used.

Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (see, eg, Chapter 4 of Kuby Immunology, 4th edition, 2000).

Transfected host cells include yeast cells and animal cells, especially mammalian cells such as CHO cells, NS0 cells, PER. human cells (such as PER. C6 or HKB-11 cells), myeloma cells, or human liver cells. Cells may be eukaryotic cells, including plant cells) as well as plant cells. In some embodiments, the transfected host cell is a mammalian cell, such as a human cell. In some embodiments, the expression host can glycosylate the antibodies of the invention with carbohydrate structures that are not themselves immunogenic, particularly in humans. In some embodiments, transfected host cells can be grown in serum-free media. In further embodiments, transfected host cells can be grown in culture in the absence of animal-derived products. Transfected host cells may be cultured to obtain cell lines.

The invention also relates to (i) preparing an immortalized B cell clone or culturing plasma cells according to the invention; (ii) converting a B cell clone or cultured plasma cell nucleic acid encoding an antibody of interest into a B cell A method for preparing one or more nucleic acid molecules encoding an antibody of interest (e.g., heavy chain and light chain genes) comprising obtaining a nucleic acid encoding the antibody of interest from cloned or cultured plasma cells. I will provide a. Furthermore, the present invention provides the following methods: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present invention; (ii) obtaining a nucleic acid from a B cell clone or cultured plasma cell encoding an antibody of interest; A method for obtaining a nucleic acid sequence encoding an antibody of interest is provided.

The invention further provides a method of preparing a nucleic acid molecule encoding an antibody of interest, comprising the step of obtaining the resulting nucleic acid from a transformed B cell clone or cultured plasma cell of the invention. Therefore, the procedure for first obtaining B cell clones or cultured plasma cells and then obtaining nucleic acids from B cell clones or cultured plasma cells can be performed by different people in different locations (e.g., in different countries). Can be done at different times.

The present invention also provides the ability to (i) obtain and/or obtain one or more nucleic acids (e.g., heavy and light chain genes) from a selected B cell clone or cultured plasma cell expressing an antibody of interest; (ii) inserting or using the nucleic acid to prepare an expression vector; (iii) transfecting a host cell capable of expressing the antibody of interest; (iv) ) culturing or subculturing the transfected host cells under conditions in which the antibody of interest is expressed; and optionally, (v) purifying the antibody of interest. Includes methods for preparing antibodies (eg, for pharmaceutical use).

The present invention also includes the step of culturing or subculturing a transfected host cell population, e.g., a stably transfected host cell population, under conditions in which antibodies of interest are expressed, and optionally , producing an antibody of interest, wherein the transfected host cell population (i) produces the selected antibody of interest produced by the B cell clone or cultured plasma cells prepared as described above; (ii) inserting the nucleic acid into an expression vector; (iii) transfecting the vector into a host cell capable of expressing the antibody of interest; and (iv) the inserted nucleic acid. A method of preparing an antibody of interest is provided, which is prepared by culturing or subculturing a transfected host cell containing the antibody to produce the antibody of interest. Therefore, the steps for first preparing recombinant host cells and then culturing them to express antibodies may be performed by different people in different locations (e.g., in different countries) and at very different times. I can do it.

(Pharmaceutical composition)
The invention also provides pharmaceutical compositions comprising one or more of the following:
(i) the antibody of the present invention or antigen-binding fragment thereof;
(ii) a nucleic acid or a combination of nucleic acids of the invention;
(iii) a vector of the invention or a combination of vectors; and/or (iv) a cell expressing an antibody of the invention or a cell containing a vector of the invention; and, optionally, a pharmaceutically acceptable excipient. agent, diluent, or carrier.

In other words, the invention also provides a pharmaceutical composition comprising an antibody according to the invention, a nucleic acid according to the invention, a vector according to the invention and/or a cell according to the invention.

Pharmaceutical compositions may also optionally include pharmaceutically acceptable carriers, diluents, and/or excipients. The carrier or excipient may facilitate administration, but should not itself induce the production of antibodies that are harmful to individuals receiving the composition. It should also be non-toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles. good. In some embodiments, pharmaceutically acceptable carriers, diluents, and/or excipients in pharmaceutical compositions of the present invention are not active ingredients with respect to MPV infection.

Pharmaceutically acceptable salts include, for example, mineral salts (such as hydrochlorides, hydrobromides, phosphates, and sulfates), or salts of organic acids (such as acetates, propionates, malonates). , and benzoates) can be used.

Pharmaceutically acceptable carriers in pharmaceutical compositions may further include liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents or pH-buffering substances may be present in such compositions. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions for ingestion by a subject.

Pharmaceutical compositions of the invention may be prepared in a variety of forms. For example, the composition may be prepared as an injectable, either as a liquid solution or suspension. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., Synagis™ and Herceptin® for reconstitution in sterile water with a preservative). similar lyophilized compositions). The composition may be prepared for topical administration, eg, as an ointment, cream, or powder. The compositions may be prepared for oral administration, eg, as tablets or capsules, as a spray, or as a syrup (optionally flavored). The compositions may be prepared for pulmonary administration, for example, as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The compositions may be prepared for nasal, aural, or ocular administration, eg, as drops. The composition may be in the form of a kit, designed so that the combined composition is reconstituted immediately prior to administration to a subject. For example, lyophilized antibodies may be provided in a kit with sterile water or sterile buffer.

In some embodiments, the (only) active ingredient in the composition is an antibody according to the invention. Therefore, it may be susceptible to degradation in the gastrointestinal tract. Thus, when the composition is administered by a route using the gastrointestinal tract, the composition may include an agent that protects the antibody from degradation, but releases the antibody once absorbed from the gastrointestinal tract.

A complete discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH of 5.5 to 8.5, and in some embodiments this may be 6 to 8, such as about 7. pH may be maintained by the use of buffers. The composition may be sterile and/or pyrogen-free. The composition may be isotonic to humans. In some embodiments, the pharmaceutical compositions of the invention are supplied in an airtight sealed container.

Within the scope of the invention are compositions that are present in several dosage forms; such forms include those forms suitable for parenteral administration, such as by injection or infusion, such as by bolus injection or Examples include, but are not limited to, continuous injection. If the product is for injection or infusion, it may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, containing suspending, preservative, stabilizing, and/or dispersing agents. It may also include preparations such as agents. Alternatively, the antibody may be in dry form for reconstitution with a suitable sterile liquid before use.

A vehicle is typically understood to be a material suitable for storing, transporting and/or administering a compound, eg a pharmaceutically active compound, in particular an antibody according to the invention. For example, a vehicle may be a physiologically acceptable liquid suitable for storing, transporting, and/or administering a pharmaceutically active compound, particularly an antibody according to the invention. Once formulated, the compositions of the invention can be administered directly to a subject. In some embodiments, the composition is adapted for administration to a mammal, eg, a human subject.

The pharmaceutical compositions of the present invention can be administered by any number of routes (oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transdermal, topical, subcutaneous, intranasal). (including, but not limited to, enteral, sublingual, intravaginal, or rectal routes). Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Optionally, the pharmaceutical composition may be prepared for oral administration (eg, for topical administration, as a tablet, capsule, etc.) or as an injection (eg, as a liquid solution or suspension). In some embodiments, the pharmaceutical composition is an injectable. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also included; eg, the pharmaceutical compositions may be in lyophilized form.

For injection, e.g., intravenous, dermal, or subcutaneous injection, or injection at the site of pain, the active ingredient is pyrogen-free and has suitable parenteral pH, isotonicity, and stability. It may be in the form of a legally acceptable aqueous solution. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, and the like. Preservatives, stabilizers, buffers, antioxidants, and/or other additives may be included as required. Whether it is an antibody, a peptide, a nucleic acid molecule, or another pharmaceutically useful compound according to the invention to be given individually, administration usually involves An "effective amount", such as a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be), is sufficient to demonstrate an individual benefit. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the invention may be provided, for example, in a prefilled syringe.

The pharmaceutical compositions of the invention as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. You can. In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, ie the transporter cargo conjugate molecule of the invention as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents may also be added.

Pharmaceutical compositions of the invention may also be administered locally, particularly when the target of treatment involves areas or organs that are readily accessible by topical application, including, for example, accessible epithelial tissue. You can. Appropriate topical formulations are readily prepared for each of these areas or organs. For topical application, the pharmaceutical composition of the invention may be formulated into a suitable ointment containing the pharmaceutical composition of the invention, in particular its components as defined above, suspended or dissolved in one or more carriers. It may be blended inside. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax, and water. Alternatively, the pharmaceutical compositions of the invention can be formulated into a suitable lotion or cream. In the context of this invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Dosage treatment may be a single dose schedule or a multiple dose schedule. In particular, the pharmaceutical composition may be provided as a single-dose product. In some embodiments, the amount of antibody in the pharmaceutical composition - particularly when provided as a single dose product - does not exceed 200 mg, such as 100 mg or 50 mg.

For a single administration, for example daily, weekly or monthly administration, the amount of antibody in the pharmaceutical composition according to the invention may not exceed 1 g or 500 mg. In some embodiments, for a single dose, the amount of antibody in a pharmaceutical composition of the invention may not exceed 200 mg, or 100 mg. For example, for a single dose, the amount of antibody in a pharmaceutical composition according to the invention may not exceed 50 mg.

Pharmaceutical compositions typically contain an "effective" amount of one or more antibodies of the invention, i.e., to treat, ameliorate, attenuate, reduce, or prevent a desired disease or condition, or to provide a detectable treatment. Contains sufficient amounts to be effective. Therapeutic effects also include reduction or attenuation of pathogenic efficacy or physical symptoms. The precise effective amount for any particular subject will depend on their size, weight, and health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective dose may generally be from about 0.005 to about 100 mg/kg, such as from about 0.0075 to about 50 mg/kg or from about 0.01 to about 10 mg/kg. . In some embodiments, an effective dose is about 0.02 to about 5 mg/kg of an antibody of the invention (e.g., the amount of antibody in the pharmaceutical composition) relative to the body weight (e.g., kg) of the individual to whom it is administered. ).

Furthermore, the pharmaceutical composition according to the invention may also contain further active ingredients, which may be further antibodies or non-antibody ingredients. Pharmaceutical compositions according to the invention may therefore contain one or more additional active ingredients.

The antibody according to the invention can be present either in the same pharmaceutical composition as the additional active ingredient, or alternatively the antibody according to the invention can be comprised by the first pharmaceutical composition and the additional active ingredient by the first pharmaceutical composition. contained by a second pharmaceutical composition different from the first pharmaceutical composition. Thus, if more than one additional active ingredient is envisaged, each additional active ingredient and the antibody according to the invention may be contained in different pharmaceutical compositions. Such different pharmaceutical compositions may be administered in combination/simultaneously or at separate times or at separate locations (eg, separate parts of the body).

Antibodies and additional active ingredients according to the invention may provide additive therapeutic effects, such as synergistic therapeutic effects. The term "synergistic" is used to describe the combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, when the combined effects of two or more agents result in "synergistic inhibition" of an activity or process, it is intended that the inhibition of the activity or process be greater than the sum of the inhibitory effects of each respective active agent. be done. The term "synergistic therapeutic effect" refers to a therapeutic effect observed in the combination of two or more treatments, where the therapeutic effect (as measured by any of a number of parameters) is observed with each individual treatment. greater than the sum of individual treatment effects.

In other embodiments, the pharmaceutical compositions of the invention may contain no additional active ingredients (in addition to the antibodies of the invention, or their respective nucleic acids, vectors, or cells, as described above).

In some embodiments, a composition of the invention may comprise an antibody of the invention, wherein the antibody accounts for at least 50% (e.g., 60%, 70%, 75%, 80% by weight of the total protein in the composition). %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more). In the compositions of the invention, the antibodies may be in purified form.

The invention also provides the steps of (i) preparing the antibodies of the invention; and (ii) mixing the purified antibodies with one or more pharmaceutically acceptable excipients, diluents, or carriers. A method of preparing a pharmaceutical composition is provided.

In other embodiments, the method of preparing a pharmaceutical composition includes the step of admixing an antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is isolated from transformed B cells of the invention or from cultured cells. This is a monoclonal antibody obtained from plasma cells.

Instead of delivering antibodies or B cells for therapeutic purposes, it is possible to deliver to a subject a nucleic acid (typically DNA) encoding a monoclonal antibody of interest derived from B cells or cultured plasma cells. , so that the nucleic acid can be expressed in situ in a subject to provide the desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

The pharmaceutical composition may also include an antimicrobial agent, particularly when packaged in a multiple dose format. They may also contain surfactants such as Tween (polysorbate) (such as Tween 80). Surfactants are generally present at low levels (eg, less than 0.01%). The compositions may also include sodium salts (eg, sodium chloride) to impart isotonicity. For example, a concentration of NaCl of 10±2 mg/ml is typical.

Furthermore, pharmaceutical compositions, especially when they are lyophilized or when they contain materials reconstituted from lyophilized materials, can contain, for example, between 15 and 30 mg/ml (e.g., 25 mg/ml). It may also contain sugar alcohols (eg, mannitol) or disaccharides (eg, sucrose or trehalose). The pH of the composition for lyophilization may be adjusted to around 5-8, or 5.5-7, or 6.1 before lyophilization.

Compositions of the invention may also include one or more immunomodulatory agents. In some embodiments, one or more of the immunomodulatory agents includes an adjuvant.

(combination of antibodies)
In a further aspect, the invention also provides a combination of separate antibodies or antigen-binding fragments thereof that binds MPV F protein (and neutralizes MPV). In particular, such combinations of antibodies may bind to different epitopes of the MPV F protein. Whether antibodies bind to the same or different epitopes can be determined, for example, using competition studies known to those skilled in the art (and as described in the Examples below).

In some embodiments, the combination of antibodies or antigen-binding fragments thereof comprises:
- an antibody or an antigen-binding fragment thereof according to the invention as described above; and - an antibody or an antigen-binding fragment thereof that binds to (a distinct epitope of) the MPV F protein.

Thus, a combination of antibodies or antigen-binding fragments thereof may include:
- (i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 5, respectively; , and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; or (ii) at least with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. heavy chain CDR1, CDR2, and CDR3 sequences having 70% sequence identity, and light chain CDR1 having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; an antibody or antigen-binding fragment thereof as described above, comprising CDR2, and CDR3 sequences; and - an antibody or antigen-binding fragment thereof that binds to (a distinct epitope of) the MPV F protein.

For example, a combination of antibodies or antigen-binding fragments thereof may include:
- an antibody or antigen-binding thereof, as described herein, comprising the heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3, or sequence variants thereof, of antibody MPF5 (MPF5_VH117D); fragment; and - an antibody or antigen-binding fragment thereof that binds to (a distinct epitope of) the MPV F protein.

More specifically, the combination of antibodies or antigen-binding fragments thereof may include:
- an antibody as described above or an antigen-binding fragment thereof, comprising the VH and VL sequences of the antibody MPF5 (MPF5_VH117D), or sequence variants thereof, as described herein; and - to (a distinct epitope of) the MPV F protein. An antibody or antigen-binding fragment thereof that binds.

Additionally, the combination of antibodies or antigen-binding fragments thereof may include:
- (i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 16, respectively; , and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 18, or (ii) at least with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. Heavy chain CDR1, CDR2, and CDR3 sequences with 70% sequence identity and light chain CDR1 with at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. , CDR2, and CDR3 sequences; and - an antibody or antigen-binding fragment thereof that binds to (a distinct epitope of) the MPV F protein.

For example, a combination of antibodies or antigen-binding fragments thereof may include:
- an antibody or antigen-binding fragment thereof as described above, comprising the heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3, or sequence variants thereof, of antibody MPE33 as described herein; and - an antibody or antigen-binding fragment thereof that binds to (a distinct epitope of) the MPV F protein.

More specifically, the combination of antibodies or antigen-binding fragments thereof may include:
- an antibody as described above or an antigen-binding fragment thereof, comprising the VH and VL sequences of antibody MPE33, or sequence variants thereof, as described herein; and - binds to (a distinct epitope of) the MPV F protein; Antibodies or antigen-binding fragments thereof.

In particular, a combination of antibodies or antigen-binding fragments thereof may include two different antibodies or antigen-binding fragments thereof of the invention as described herein. Such antibodies or antigen-binding fragments thereof of the invention may bind different epitopes of the MPV F protein.

Thus, a combination of antibodies or antigen-binding fragments thereof may include:
- (i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 5, respectively; , and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; or (ii) at least with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Heavy chain CDR1, CDR2, and CDR3 sequences with 70% sequence identity and light chain CDR1 with at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. , CDR2, and CDR3 sequences; and -(iii) having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. or ( iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 17, respectively; An antibody or antigen-binding fragment thereof as described above, comprising a light chain CDR1, CDR2, and CDR3 sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 18.

For example, a combination of antibodies or antigen-binding fragments thereof may include:
- an antibody or antigen-binding thereof, as described herein, comprising the heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3, or sequence variants thereof, of antibody MPF5 (MPF5_VH117D); fragments; and - the heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3 of antibody MPE33, or an antibody described herein or comprising a sequence variant described herein. its antigen-binding fragment.

More specifically, the combination of antibodies or antigen-binding fragments thereof may include:
- an antibody as described above or an antigen-binding fragment thereof comprising the VH and VL sequences of the antibody MPF5 (MPF5_VH117D), or sequence variants thereof, as described herein; and - of the antibody MPE33, as described herein. An antibody or antigen-binding fragment thereof as described above, comprising VH and VL sequences, or sequence variants thereof.

For example, heavy chain CDR1 as set forth in SEQ ID NO: 1, heavy chain CDR2 as set forth in SEQ ID NO: 2, and heavy chain CDR3 as set forth in SEQ ID NO: 3 or 10; and light chain CDR1 as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or The antibody or antigen-binding fragment thereof comprises the light chain CDR2 set forth in SEQ ID NO: 6 and the light chain CDR3 set forth in SEQ ID NO: 7; heavy chain CDR1 as set forth in SEQ ID NO: 13, and heavy chain CDR3 as set forth in SEQ ID NO: 14; and light chain CDR1 as set forth in SEQ ID NO: 15, light chain CDR1 as set forth in SEQ ID NO: 16 or 17; may be combined with an antibody or antigen-binding fragment thereof comprising chain CDR2, and light chain CDR3 as set forth in SEQ ID NO:18.

More specifically, the antibody or antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO: 8 or 11 and the VL set forth in SEQ ID NO: 9 comprises SEQ ID NO: 19, 22, 24, 26, 28, 30, 32, or an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO: 34 and the VL set forth in SEQ ID NO: 20.

In the combination of antibodies or antigen-binding fragments thereof of the present invention, the antibodies or antigen-binding fragments thereof may be, for example, proteins (antibodies), nucleic acids (encoding the above antibodies), vectors (comprising the above nucleic acids), cells (containing the above antibodies). may be provided in any of the forms described above. Accordingly, the invention provides combinations comprising (i) two different antibodies of the invention, or antigen-binding fragments thereof;
(ii) two different nucleic acids (or combinations of nucleic acids) of the invention;
(iii) two different vectors (or combinations of vectors) of the invention; or (iv) two different vectors (or combinations of vectors) of the invention; separate cells.

The combination of antibodies of the invention or antigen-binding fragments thereof (in any of the forms described above, such as antibodies (proteins)) may be included in the same composition or in separate compositions (e.g., pharmaceutical compositions as described above). is understood. The invention therefore also provides pharmaceutical compositions comprising two different antibodies or antigen-binding fragments of the invention as described above.

(Medical treatment and other uses)
In a further aspect, the invention provides antibodies according to the invention or antigen-binding fragments thereof, nucleic acid molecules (or combinations of nucleic acid molecules) according to the invention, vectors (or combinations of vectors) according to the invention, cells according to the invention. , or the use of the pharmaceutical composition according to the present invention as a medicine. In particular, the antibody according to the present invention or an antigen-binding fragment thereof, the nucleic acid molecule according to the present invention (or a combination of nucleic acid molecules), the vector according to the present invention (or a combination of vectors), the cell according to the present invention, the The pharmaceutical composition or combination of antibodies or antigen-binding fragments thereof according to the invention may be used in the prevention and/or treatment of MPV infection; or (ii) in the diagnosis of MPV infection.

Therefore, the present invention also provides a therapeutically effective amount of an antibody according to the present invention or an antigen-binding fragment thereof, a nucleic acid molecule according to the present invention (or a combination of nucleic acid molecules), a vector according to the present invention (or a combination of vectors) according to the present invention, The present invention provides a method for improving or reducing MPV infection, or reducing the risk of MPV infection, which comprises administering a cell according to the present invention or a pharmaceutical composition according to the present invention to a subject in need thereof. Furthermore, the present invention covers the antibody or antigen-binding fragment thereof according to the present invention, the nucleic acid molecule according to the present invention (or a combination of nucleic acid molecules), the vector according to the present invention (or a combination of vectors), the cell according to the present invention, the present invention Also provided is the use of a pharmaceutical composition according to the invention or a combination of antibodies or antigen-binding fragments thereof according to the invention in the manufacture of a medicament for the prevention, treatment or attenuation of MPV infection.

Prevention of MPV infection is particularly important if the subject has not been diagnosed with MPV (either no diagnosis was made or the diagnosis was negative) and/or the subject has symptoms of MPV infection. Refers to a preventive situation in which no In a therapeutic setting, by contrast, the subject is typically diagnosed with and/or exhibiting symptoms of MPV infection. Of note, the terms "treatment" and "therapy"/"therapeutic" of MPV infection refer to (complete) cure and attenuation/of MPV infection and/or associated symptoms. Including reduction.

In some embodiments, the subject may be human. One method of testing the effectiveness of therapeutic treatment involves monitoring disease symptoms following administration of the compositions of the invention. Treatment may be a single dose schedule or a multiple dose schedule. In one embodiment, antibodies, antibody fragments, nucleic acids, vectors, cells, or compositions of the invention are administered to a subject in need of such treatment. Such subjects include, but are not limited to, subjects who are particularly at risk or susceptible to MPV infection, including, for example, immunocompromised subjects.

The antibodies and fragments thereof described in the invention may also be used for the diagnosis of MPV infection. A method of diagnosis may include contacting an antibody with a sample. Such samples include, for example, the nasal cavity, sinuses, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart, ovaries, pituitary gland, adrenal glands, thyroid, brain, skin, or blood ( An isolated tissue sample obtained from the subject (such as plasma or serum) may be isolated from the subject. For example, an antibody or antigen-binding fragment thereof may be contacted with an (isolated) blood sample (eg, whole blood, plasma, or serum). Diagnostic methods may also include detection of antigen/antibody complexes, particularly after contacting the antibody with a sample. Such a detection step may typically be performed on the bench, ie, without any contact with the human or animal body. Examples of detection methods are well known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay). Diagnosis may therefore be carried out in vitro (and the step of in vitro detection of antigen/antibody complexes), for example by using the isolated samples described above. Antibodies or antigen-binding fragments thereof may therefore be used in the (in vitro) diagnosis of MPV infection.

The antibodies of the invention or antigen-binding fragments thereof may therefore be used in (in vitro) methods for detecting MPV antigens. Similarly, antibodies of the invention or antigen-binding fragments thereof may be used in (in vitro) methods for binding MPV target proteins/antigens, such as MPV F protein or antigenic fragments or variants thereof. Because of their specificity, the antibodies of the invention or antigen-binding fragments thereof recognize the MPV F protein, particularly its prefusion conformation. To detect the MPV antigen, the antibody may be contacted with the (isolated) sample (ie, the sample to be tested for the presence of the antigen). The specific binding of an antibody to its antigen (MPV F protein) forms an antibody/antigen complex, which can be easily detected by methods known in the art.

Such detection methods may be used in an (in vitro) diagnostic context (using samples isolated from the human or animal body), but also in other (e.g. production/manufacturing) contexts such as vaccine samples. It may also be used to test samples. Accordingly, the antibodies, antibody fragments or variants thereof according to the invention may also be used in non-therapeutic/non-diagnostic contexts (eg in vaccine development or manufacture). Therefore, the present invention also provides a method for testing vaccines, in particular when the antigen (i.e. the desired antigen contained in the vaccine) is properly produced and/or folded (and/or in the correct conformation). Provided is the use of the antibodies of the invention or antigen-binding fragments thereof to test whether Therefore, antibodies may be used to monitor vaccine production with desired immunogenicity. For this purpose, the antibody may be contacted with a vaccine, eg as described above. Accordingly, the present invention also provides a method for testing an anti-MPV vaccine, wherein the vaccine is contacted with an antibody or antigen-binding fragment thereof and, optionally, the presence of an antibody/antigen complex is determined. In addition, the present invention also provides anti-MPV vaccines by testing whether the vaccine contains the desired antigen, e.g., the F protein of MPV (e.g., in a prefusion conformation), or a fragment or variant thereof. Includes the use of antibodies of the invention or antigen-binding fragments thereof to monitor the quality of vaccines. More specifically, antibodies may be used to examine the conformation of an antigen or its epitope in a vaccine. Since the antibodies of the invention specifically bind to prefusion F protein, detection of a significant amount of antibody/antigen complex in a sample means that the sample contains MPV F protein primarily in the prefusion conformation. You may. Additionally, modified forms of the antigen can also be tested using antibodies of the invention, such as fragments and variants of the MPV F protein that may be useful in vaccines.

[Brief explanation of the drawing]
In the following a brief description of the attached drawings is given. The drawings are intended to explain the invention in more detail. However, they are in no way intended to limit the content of the invention.

FIG. 1 shows the binding properties of MPE33 and MPF5 antibodies to hMPV F protein in prefusion and postfusion conformations compared to reference antibody MPE8 for Example 1.

FIG. 2 shows, for Example 2, four different hMPV strains A1/6621 (MPV A1), A2/VR8938 (MPV A2), B1/VR4702 (MPV B1) with antibodies MPE33 and MPF5 compared to reference antibody MPE8v3. ), and B2/3817 (MPV B2).

FIG. 3 shows, for Example 2, four different hMPV strains A1/6621 (MPV A1), A2/VR8938 (MPV A2), B1/VR4702 (MPV B1) with antibodies MPE33 and MPF5 compared to reference antibody MPE8v3. ), and the EC50 required for neutralization of B2/3817 (MPV B2).

FIG. 4 shows the binding affinity and EC50 values of antibodies MPF5_VH117D (MPF5), MPF5_VH117H, MPE33, and comparative antibody MPE8v3 for the prefusion F protein of MPV for Example 3.

FIG. 5 shows the VH and VL (“VK”) sequences of antibodies MPF5_VH117D (MPF5) and MPF5_VH117H. The box indicates the position of the substitution at amino acid position VH117.

FIG. 6 shows the binding affinity and EC50 values of antibody MPE33 and its variants MPE33_S36A, MPE33_N34Q, MPE33_N34S, MPE33_C38S, MPE33_C38A, MPE33_C38Y, and MPE33_N34S_C38Y for prefusion F protein of MPV for Example 4. shows.

Figure 7 shows the VH and VL ("VK") sequences of antibody MPE33. Boxes indicate the positions of substitutions at amino acid positions N34, S36, and C38.

FIG. 8 shows, for Example 5, antibodies MPE33, MPE33_S36A, MPE33_N34Q, MPE33_N34S, against cell-bound F antigen of hMPV strains MPV_NL/1/99_F0-TM (upper row) and HMPV_Yokohama/JPN (P8527) 2016 (interrupted); Binding of MPE33_C38S, MPE33_C38A, MPE33_C38Y, MPE33_N34S_C38Y, MPF5_VH117D (MPF5), MPF5_VH117H, and comparison antibody MPE8 is shown. At the bottom, mock transfection (control) is shown.

FIG. 9 shows the results of a competition study of antibodies MPF5, MPE33, and MPE8 for binding to MPV F protein for Example 6. The data show competition between the same antibodies as expected (MPF5 vs. MPF5; MPE33 vs. MPE33; MPE8 vs. MPE8). Additionally, panels MPF5 vs. MPE8 and panels MPE8 vs. MPF5 demonstrate competition between MPF5 and MPE8. However, no competition was observed between MPE33 and MPE8 or between MPE33 and MPF5.

〔Example〕
Below, specific examples are presented that illustrate various embodiments and aspects of the invention. However, the invention is not limited in scope by the particular embodiments described herein. The following Preparations and Examples are given to enable those skilled in the art to more clearly understand and practice the invention. However, the invention is not limited in scope by the illustrated embodiments, which are intended only as illustrations of single aspects of the invention, and functionally equivalent methods are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying drawings, and the following examples. All such modifications are within the scope of the appended claims.

(Example 1: Identification and characterization of human monoclonal antibodies MPF5 and MPE33)
Human monoclonal antibodies MPF5 (also called "MPF5_VH117D") and MPE33 against MPV were isolated from human patients (see Traggiai E. et al., 2004, Nat Med 10(8): 871-5). The antibody can be expressed by the nucleotide and amino acid sequences of its variable regions (MPF5 VH: SEQ ID NO: 8, MPF5 VL: SEQ ID NO: 9; MPE33 VH: SEQ ID NO: 19, MPE33 VL: SEQ ID NO: 20) and the complementarity determining region therein. (CRD) (MPF5: SEQ ID NO: 1-5 and 7, or 1-4, 6 and 7, respectively; MPE33: SEQ ID NO: 12-16 and 18, or 12-15, 17 and 18, respectively) characterized by. The VH and VL genes of MPF5 and MPE33 were cloned into an IgG1 expression vector, and recombinant antibodies were generated by transient transfection of 293Freestyle cells (293F). Supernatants were collected from transfected cells and IgG was affinity purified by protein A chromatography. Therefore, MPF5 and MPE33 are fully human monoclonal antibodies of the IgG1 type with the CDR, VH, and VL sequences described herein.

To test the binding of MPF5 and MPE33 to hMPV F protein, an ELISA assay was developed essentially as per WO2016/103238A1 to test the binding affinity of antibodies to hMPV F protein in pre-fusion versus post-fusion conformations. I went as described. For comparison, the prior art antibody MPE8 (Corti et al., 2013, Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501: 439-443) was also tested in this experiment.

Briefly, Maxisorp ELISA plates were loaded with conformationally stabilized pre- and post-fusion F protein antigen (F protein from CAN97-83 MPV strain, 1 μg/ml, 25 μl/well in PBS pH 7). Both were coated overnight. After three washes with PBS/Tween 0.01% (PBST), test antibodies were added starting at 10 μg/ml, titrated threefold to 11 points, and incubated for 2 hours at room temperature. Plates were then washed four times in PBST, and alkaline phosphatase-labeled goat anti-human IgG polyclonal antibody (SouthernBiotech, 2 μg/ml, 25 μl/well) was dispensed and further incubated for 1 hour at RT. After four washes with PBST, plates were developed by adding 50 μl/well of AP substrate (pNPP, Sigma) in carbonate buffer and read at 405 nm after 45 minutes.

The results are shown in Figure 1. All antibodies tested (MPF5, MPE33, and MPE8) showed high binding affinity for the MPV prefusion F protein but not for the MPV postfusion F protein. There wasn't. Therefore, antibodies MPF5, MPE33, and MPE8 are specific for the prefusion F protein of MPV. Antibodies MPF5 and MPE33 of the invention exhibited lower EC50 values and therefore higher binding affinity for the prefusion F protein of MPV compared to the prior art antibody MPE8.

Example 2: MPF5 and MPE33 effectively neutralize various strains of MPV
Neutralization of various different strains of MPV was then evaluated using the antibodies MPF5 and MPE33 of the invention, and the comparative antibody MPE8v3, which contains the N113S mutation in the heavy chain variable region to remove glycosylation sites. It differs from the control antibody MPE8 used in Example 1 in that it contains the following.

Briefly, culture supernatants containing antibodies were analyzed using a microneutralization assay based on infection of LLC-MK2 cells with hMPV strains A1/6621, A2/VR8938, B1/VR4702, and B2/3817. The neat supernatant was 0.239*10 ⁶ TCID50/ml (A1/6621), 0.0959*10 ⁶ TCID50/ml (A2/VR8938), and 0.33*10 ⁶ TCID50/ml (B1/VR4702), respectively. ) and 0.0959*10 ⁶ TCID50/ml (B2/3817) of virus for 1 hour at room temperature, then LLC-MK2 target cells were added and incubated for 14 days, respectively. Viable cells were detected using WST-1 reagent (Roche). EC50 was determined using the microneutralization assay described above with 100 TCID ₅₀ of virus, and viral infection was determined on day 6 or 7 by indirect immunofluorescence using an automated pathway 855 analyzer (BD). did. EC50 values were calculated by interpolation of the neutralization curve fitting a 4-parameter non-linear regression with variable slope.

The results are shown in FIGS. 2 and 3. All antibodies effectively neutralized the four different MPV strains tested. Specifically, MPE33 and MPF5 showed significantly better neutralizing efficacy against the MPV B2 strain compared to the reference mAb MPE8v3.

(Example 3: CDRH3 variants of MPF5 exhibit similarly high binding affinity for MPV prefusion F protein)
Next, an MPF5 mutant antibody MPF5_VH117H was produced, which differs from MPF5 in that its CDRH3 sequence is according to SEQ ID NO: 10 and its VH sequence is according to SEQ ID NO: 11. Differences in the MPF5 heavy chain sequence and CDRH3 are shown in FIG. 5.

The binding affinity of antibodies MPF5_VH117D (MPF5), MPF5_VH117H, MPE8v3, and MPE33 to the prefusion F protein of MPV was tested in the ELISA assay described in Example 1.

The results are shown in Figure 4. Although all antibodies tested specifically bound to the prefusion F protein of MPV, the binding affinities of the antibodies of the invention MPF5_VH117D (MPF5), MPF5_VH117H, and MPE33 were significantly greater than those of the comparison antibody MPE8v3. it was high. The binding affinities of antibodies MPF5_VH117D (MPF5) and MPF5_VH117H were similarly high, indicating that the CDRH3 mutation did not impair the binding affinity of MPF5 to the prefusion F protein of MPV.

(Example 4: MPE33 CDRH1 variant shows similarly high binding affinity for MPV prefusion F protein)
Next, we generated the following mutant antibodies of MPE33 that differed from MPE33 only in the indicated CDRH1 sequences:

The heavy chain sequence of MPE33 and the positions of the mutant amino acids in CDRH3 are shown in FIG.

The binding affinity of the antibody MPE33 and its variant antibodies described above for the prefusion F protein of MPV was tested in the ELISA assay described in Example 1.

The results are shown in FIG. All tested antibodies MPE33, MPE33_S36A, MPE33_N34Q, MPE33_N34S, MPE33_C38S, MPE33_C38A, MPE33_C38Y, and MPE33_N34S_C38Y specifically bound to the prefusion F protein of MPV with similarly high binding affinity. The binding affinities of all variant antibodies were even slightly higher than that of MPE33 to the prefusion F protein of MPV, and various CDRH1 mutations lowered the binding affinity of MPE33 to the prefusion F protein of MPV. It shows that there was no damage.

(Example 5: Binding to cell-bound F antigen)
The binding of all exemplified antibodies of the invention as described in the Examples above as well as the comparative antibody MPE8 was then tested.

For this purpose, Expi293 cells were transfected with MPV F protein (MPV_NL/1/99_F0-TM (AY304361) and HMPV_Yokohama/JPN (P8527) 2016). HMPV_Yokohama/JPN (P8527) 2016 carries the D280N mutation. Briefly, 10 μg of plasmid DNA was diluted in 0.5 mL of Opti-Mem I medium (Gibco, cat. #31985-047) and 30 μl of PEI Max transfection reagent (40 kD, cat. # POL24765-1, 0.5 mL of Opti-Mem containing Polysciences) and incubated for 20 minutes at room temperature (RT). The transfection mix was then added to a culture flask containing 80 ml of expression medium (GIBCO, cat #A14351-02) while growing Expi293 cells (3 x ¹⁰ cells/ml) and incubated at 37 °C under agitation. Incubated for 3 days. Cells were then harvested, fixed with 4% formaldehyde for 20 min on ice, permeabilized with 0.5% saponin, 1% FBS in PBS for 20 min on ice, and incubated with antibodies MPE33, MPE33_S36A, MPE33_N34Q, MPE33_N34S, MPE33_C38S, MPE33_C38A, MPE33_C38Y, and MPE33_N34S_C38Y, MPF5_VH117D (MPF5), MPF5_VH117H, and comparison antibody MPE8 (5 μg/ml in permeabilization buffer, 60 min at 4°C) were stained. Binding is performed by staining cells with AF647 goat anti-human IgG, Fcγ fragment-specific secondary antibody (Jackson, 109-606-098, 1 μg/ml 30 min, on ice) and acquiring cells with a flow cytometer. It was made clear by this.

The results are shown in FIG. 8, with MPV_NL/1/99_F0-TM in the upper row, HMPV_Yokohama/JPN (P8527) 2016 in the middle row, and mock transfection in the lower row. These data show that MPE33 and MPF5 and all their variants bind equally well to the F protein of both hMPV strains B1 (NL1/1/99) and B2 (Yokohama), whereas the B2 strain ( D280N mutation) is shown to generate a virus escape variant of the MPE8 antibody.

Example 6: Competition study of antibodies MPE33, MPF5, and MPE8
Binding/competition studies were performed to identify whether antibodies MPE33 and MPF5 bind to the same or different epitopes on the MPV F protein as comparison antibody MPE8.

To this end, the competition of antibodies MPE33, MPF5, and MPE8 was evaluated by biolayer interference using OCTET RED96 (ForteBio). Briefly, APS sensors were loaded/coated with 5 μg/ml MPV F protein in PBS for 10 minutes. Sequentially, the sensor was blocked with 1 mg/ml BSA in PBS (blocking buffer) for 5 minutes. mAb association was performed by transferring the sensor to two consecutive wells (7 min each) containing the first and second mAb at 30 ug/ml each in blocking buffer. All steps were carried out at 30° C. with constant mixing at 1000 rpm.

The results are shown in FIG. Data demonstrate that MPF5 competes with MPE8 and show that both antibodies bind to the same or overlapping epitopes on the MPV F protein. However, MPE33 did not compete with either MPF5 or MPE8, indicating that MPE33 binds to a different epitope on the MPV F protein compared to MPF5 and MPE8.

FIG. 1 shows the binding properties of MPE33 and MPF5 antibodies to hMPV F protein in prefusion and postfusion conformations compared to reference antibody MPE8 for Example 1. FIG. 2 shows, for Example 2, four different hMPV strains A1/6621 (MPV A1), A2/VR8938 (MPV A2), B1/VR4702 (MPV B1) with antibodies MPE33 and MPF5 compared to reference antibody MPE8v3. ), and B2/3817 (MPV B2). FIG. 3 shows, for Example 2, four different hMPV strains A1/6621 (MPV A1), A2/VR8938 (MPV A2), B1/VR4702 (MPV B1) with antibodies MPE33 and MPF5 compared to reference antibody MPE8v3. ), and the EC50 required for neutralization of B2/3817 (MPV B2). FIG. 4 shows the binding affinity and EC50 values of antibodies MPF5_VH117D (MPF5), MPF5_VH117H, MPE33, and comparative antibody MPE8v3 for the prefusion F protein of MPV for Example 3. FIG. 5 shows the VH and VL (“VK”) sequences of antibodies MPF5_VH117D (MPF5) and MPF5_VH117H. The box indicates the position of the substitution at amino acid position VH117. FIG. 6 shows the binding affinity and EC50 values of antibody MPE33 and its variants MPE33_S36A, MPE33_N34Q, MPE33_N34S, MPE33_C38S, MPE33_C38A, MPE33_C38Y, and MPE33_N34S_C38Y for prefusion F protein of MPV for Example 4. shows. Figure 7 shows the VH and VL ("VK") sequences of antibody MPE33. Boxes indicate the positions of substitutions at amino acid positions N34, S36, and C38. FIG. 8 shows, for Example 5, antibodies MPE33, MPE33_S36A, MPE33_N34Q, MPE33_N34S, against cell-bound F antigen of hMPV strains MPV_NL/1/99_F0-TM (upper row) and HMPV_Yokohama/JPN (P8527) 2016 (interrupted); Binding of MPE33_C38S, MPE33_C38A, MPE33_C38Y, MPE33_N34S_C38Y, MPF5_VH117D (MPF5), MPF5_VH117H, and comparison antibody MPE8 is shown. At the bottom, mock transfection (control) is shown. FIG. 9 shows the results of a competition study of antibodies MPF5, MPE33, and MPE8 for binding to MPV F protein for Example 6. The data show competition between the same antibodies as expected (MPF5 vs. MPF5; MPE33 vs. MPE33; MPE8 vs. MPE8). Additionally, panels MPF5 vs. MPE8 and panels MPE8 vs. MPF5 demonstrate competition between MPF5 and MPE8. However, no competition was observed between MPE33 and MPE8 or between MPE33 and MPF5.

Claims (72)

An antibody or antigen-binding fragment thereof that binds to the F protein of metapneumovirus (MPV). The antibody or antigen-binding fragment thereof is
(i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 5, respectively; a light chain CDR1, CDR2, and CDR3 sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; or
(ii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 6, respectively; light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; or (iii) at least 70% with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, respectively. CDR3 sequences; or (iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 15, respectively; 17, and light chain CDR1, CDR2, and CDR3 sequences having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 18. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to prefusion F protein of MPV. Claim 3, wherein at least 100 times higher concentration of the antibody or antigen-binding fragment thereof is required for 50% antibody binding to post-fusion F protein of MPV than for 50% antibody binding to pre-fusion F protein of MPV. The antibody or antigen-binding fragment thereof described. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof neutralizes MPV infection. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof specifically binds to F proteins of MPV subgroups A1, A2, B1, and B2. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof neutralizes infection of MPV subgroups A1, A2, B1, and B2. The antibody or antigen-binding fragment thereof is
(i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 5, respectively; a light chain CDR1, CDR2, and CDR3 sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7; or
(ii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 6, respectively; light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7; or (iii) at least 80% with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. and light chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, respectively. CDR3 sequences; or (iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 80% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 15, respectively; No. 17, and a light chain CDR1, CDR2, and CDR3 sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID No. 18, or its antigen. Combined fragment. The antibody or antigen-binding fragment thereof is
(i) heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 5, respectively; a light chain CDR1, CDR2, and CDR3 sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7; or
(ii) heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and SEQ ID NO: 4, SEQ ID NO: 6, and light chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:7; or (iii) at least 90% with the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively. and light chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 18, respectively. CDR3 sequences; or (iv) heavy chain CDR1, CDR2, and CDR3 sequences having at least 90% sequence identity with the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and SEQ ID NO: 15, SEQ ID NO: 15, respectively; No. 17, and a light chain CDR1, CDR2, and CDR3 sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID No. 18, or an antigen thereof according to any one of claims 1 to 8. Combined fragment. The antibody or antigen-binding fragment thereof is
- heavy chain CDR1 sequence according to SEQ ID NO: 1;
- heavy chain CDR2 sequence according to SEQ ID NO: 2;
- a heavy chain CDR3 sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3;
- light chain CDR1 sequence according to SEQ ID NO: 4;
- the light chain CDR2 sequence according to SEQ ID NO: 5 or 6; and - the light chain CDR3 sequence according to SEQ ID NO: 7. 11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the C-terminal Asp residue of SEQ ID NO: 3 is substituted; optionally, it may be substituted with another polar amino acid. 12. The antibody or antigen-binding fragment thereof according to claim 10 or 11, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain CDR3 sequence set forth in SEQ ID NO: 3 or 10. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 1;
- heavy chain CDR2 sequence according to SEQ ID NO: 2;
- heavy chain CDR3 sequence according to SEQ ID NO: 3;
- light chain CDR1 sequence according to SEQ ID NO: 4;
- a light chain CDR2 sequence according to SEQ ID NO: 5 or 6; and - a light chain CDR3 sequence according to SEQ ID NO: 7. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 1;
- heavy chain CDR2 sequence according to SEQ ID NO: 2;
- heavy chain CDR3 sequence according to SEQ ID NO: 10;
- light chain CDR1 sequence according to SEQ ID NO: 4;
- a light chain CDR2 sequence according to SEQ ID NO: 5 or 6; and - a light chain CDR3 sequence according to SEQ ID NO: 7. The antibody or antigen-binding fragment thereof:
- a heavy chain CDR1 sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 12;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. 16. The antibody or its antibody according to claim 15, wherein one or more of the heavy chain variable region amino acid residues N34, S36 and C38 (corresponding to N6, S8 and C10 in SEQ ID NO: 12, respectively) are substituted. Antigen-binding fragment. - N34 (corresponding to N6 in SEQ ID NO: 12) is replaced with Gln (Q) or Ser (S);
-S36 (corresponding to S8 in SEQ ID NO: 12) is replaced with Ala(A); and/or -C38 (corresponding to C10 in SEQ ID NO: 12) is replaced with Ser(S), Ala(A) or Tyr(Y), the antibody or antigen-binding fragment thereof according to claim 16. 18. The antibody or antigen-binding fragment thereof according to any one of claims 15 to 17, wherein the heavy chain CDR1 sequence differs from SEQ ID NO: 12 by a single or exactly two amino acid substitutions from SEQ ID NO: 12. Any one of claims 15 to 18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 sequence according to any one of SEQ ID NOs: 12, 21, 23, 25, 27, 29, 31 and 33. The antibody or antigen-binding fragment thereof described in Section 1. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 12;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 21;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 23;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 25;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 27;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 29;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 31;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof:
- heavy chain CDR1 sequence according to SEQ ID NO: 33;
- heavy chain CDR2 sequence according to SEQ ID NO: 13;
- heavy chain CDR3 sequence according to SEQ ID NO: 14;
- light chain CDR1 sequence according to SEQ ID NO: 15;
- the light chain CDR2 sequence according to SEQ ID NO: 16 or 17; and - the light chain CDR3 sequence according to SEQ ID NO: 18. The antibody or antigen-binding fragment thereof is
(i) a heavy chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 9; or (ii) the sequence A heavy chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to any one of the above. The antibody or antigen-binding fragment thereof is
(i) a heavy chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 9; or (ii) the sequence A heavy chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to any one of the above. The antibody or antigen-binding fragment thereof is
(i) a heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 9; or (ii) a sequence A heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to any one of the above. The antibody or antigen-binding fragment thereof is
(i) a heavy chain variable region comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 9; or (ii) a sequence 19, and a light chain variable region comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to any one of the above. The antibody or antigen-binding fragment thereof is
(i) a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 9; or (ii) a sequence A heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to any one of the above. The antibody or antigen-binding fragment thereof is

(i) a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 9; or (ii) a sequence A heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 20. The antibody or antigen-binding fragment thereof according to any one of the above. 34. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8 and a light chain variable region set forth in SEQ ID NO: 9. Antibodies or antigen-binding fragments thereof. 35. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region set forth in SEQ ID NO: 9. Antibodies or antigen-binding fragments thereof. 36. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. 37. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. 38. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. 39. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 26 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. 40. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28 and a light chain variable region set forth in SEQ ID NO:20. Antibodies or antigen-binding fragments thereof. 41. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. 42. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. 43. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable region set forth in SEQ ID NO: 20. Antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 43, wherein the antibody or antigen-binding fragment thereof is a human antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 44, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody. 46. An antibody according to any one of claims 1 to 45, wherein said antibody comprises an Fc portion. Antibody according to any one of claims 1 to 46, wherein the antibody is of the IgG type. 48. The antibody of claim 47, wherein said antibody is of type IgG1. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 48, wherein the antibody or antigen-binding fragment thereof is purified. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 49, wherein the antibody or antigen-binding fragment thereof is a single chain antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 50, wherein the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv or scFv. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 51 for use as a medicament. 53. An antibody or antigen-binding fragment thereof according to claim 52 for use in the prevention or treatment of MPV infection. A nucleic acid molecule comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 51. 55. The nucleic acid molecule of claim 54, wherein said polynucleotide encoding said antibody or antigen binding fragment thereof is codon optimized. or at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 56. A nucleic acid molecule according to claim 54 or 55, comprising sequence variants thereof having 96%, at least 97%, at least 98% or at least 99% sequence identity. a combination of a first and a second nucleic acid molecule,
The first nucleic acid molecule comprises a polynucleotide encoding the heavy chain of an antibody according to any one of claims 1 to 51 or an antigen-binding fragment thereof; A combination of first and second nucleic acid molecules comprising a polynucleotide encoding the corresponding light chain of an antibody or the same antigen-binding fragment thereof. 58. The combination of nucleic acid molecules according to claim 57, wherein one or both of the polynucleotides encoding the antibody heavy and/or light chain or antigen-binding fragment thereof are codon-optimized. A combination of nucleic acid molecules comprising a nucleic acid sequence according to any one of SEQ ID NOs: 38-55; or at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 59. A combination of nucleic acid molecules according to claim 57 or 58, comprising sequence variants thereof having 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. . a combination of a first and a second nucleic acid molecule,
(i) said first nucleic acid molecule comprises a polynucleotide encoding an antibody heavy chain or antigen-binding fragment thereof, said polynucleotide comprising (a) the nucleotide sequence set forth in SEQ ID NOs: 38, 39, and 40; or ( b) comprises the nucleotide sequences set forth in SEQ ID NOs: 47, 48 and 49; and (ii) said second nucleic acid molecule comprises a polynucleotide encoding an antibody light chain or antigen-binding fragment thereof, said polynucleotide comprising: , (c) the nucleotide sequences set forth in SEQ ID NOs: 41, 42 (or 43) and 44; or (d) the nucleotide sequences set forth in SEQ ID NOs: 50, 51 (or 52) and 53. A combination of nucleic acid molecules. A vector comprising a nucleic acid molecule according to any one of claims 54 to 56 or a combination of nucleic acid molecules according to any one of claims 57 to 60. A combination of a first and a second vector, wherein the first vector comprises the first nucleic acid molecule according to any one of claims 57 to 60, and the second vector comprises the first nucleic acid molecule according to any one of claims 57 to 60. A combination of a first and second vector comprising a nucleic acid molecule corresponding to the second nucleic acid molecule according to item 1. A cell expressing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 51 or comprising a vector according to claim 61 or a combination of vectors according to claim 62. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, the nucleic acid according to any one of claims 54 to 56, the nucleic acid according to any one of claims 57 to 60. a combination of a vector according to claim 61, a combination of vectors according to claim 62, or a cell according to claim 63, and, optionally, a pharmaceutically acceptable excipient, diluent or carrier, A pharmaceutical composition comprising. An antibody according to any one of claims 1 to 51 or an antigen-binding fragment thereof according to any one of claims 54 to 56, for use as a medicament or optionally in the prevention or treatment of MPV infection. a nucleic acid according to any one of claims 57 to 60, a vector according to claim 61, a vector combination according to claim 62, a cell according to claim 63, or a cell according to claim 64. The pharmaceutical composition described in . Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 51 in the (in vitro) diagnosis of MPV infection. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 51 in a method for detecting an MPV antigen. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 51 for monitoring the quality of an anti-MPV vaccine by testing the antigen of the vaccine. 69. The use according to claim 68, wherein the conformation of the vaccine antigen or epitope thereof is examined. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, the nucleic acid according to any one of claims 54 to 56, the nucleic acid according to any one of claims 57 to 60. combination, a vector according to claim 61, a combination of vectors according to claim 62, a cell according to claim 63, or a pharmaceutical composition according to claim 64, for the prevention, treatment or attenuation of MPV infection. Use in the manufacture of medicines. A therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, a nucleic acid according to any one of claims 54 to 56, or a nucleic acid according to any one of claims 57 to 60. A combination of nucleic acids according to claim 61, a vector combination according to claim 62, a cell according to claim 63, or a pharmaceutical composition according to claim 64 in need thereof. A method of reducing MPV infection, or reducing the risk of MPV infection, comprising administering to a subject. 52. A method for testing an anti-MPV vaccine, wherein said vaccine is contacted with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 51, optionally comprising the step of testing an anti-MPV vaccine for the presence of an antibody/antigen complex. The method by which is determined.

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